Suppression of tla1 gene expression for improved photosynthetic productivity

ABSTRACT

The invention provides method and compositions to minimize the chlorophyll antenna size of photosynthesis by decreasing TLA1 gene expression, thereby improving solar conversion efficiencies and photosynthetic productivity in plants, e.g., green microalgae, under bright sunlight conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/782,124, filed May 18, 2010, which is a divisional application ofU.S. application Ser. No. 11/423,620, filed Jun. 12, 2006, now U.S. Pat.No. 7,745,696, the content of which applications are hereby incorporatedby reference in their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant (Contract)Nos. DE-FC36-00GO10536 and DE-FG36-05GO15041 awarded by the UnitedStates Department of Energy. The Government has certain rights in thisinvention.

REFERENCE TO SEQUENCE LISTING

This application includes a Sequence Listing as a text file named“79438-899342-SEQLISTING.TXT” created Feb. 4, 2014, and containing29,245 bytes, machine format IBM-PC, MS-Windows operating system, whichis hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Oxygenic photosynthesis depends on the absorption of sunlight byauxiliary light-harvesting pigments, which are incorporated within theholocomplexes of photosystem-I and photosystem-II. In each photosystem(PS), sizable arrays of chlorophylls and other accessory pigments (e.g.,carotenoids) act cooperatively as antennae for the collection of lightenergy and as a conducting medium for excitation migration toward aphotochemical reaction center (see, e.g., Emerson & Arnold, J GenPhysiol 15: 391-420, 1932; Emerson & Arnold, J Gen Physiol 16: 191-205,1933; Gaffron & Wohl, Naturwissenschaften 24: 81-90, 1936; Melis, In,Oxygenic Photosynthesis: The Light Reactions” (D R Ort, C F Yocum, eds),Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 523-538,1996). Organized as distinct pigment-protein complexes and containedwithin PSI and PSII, these light-harvesting antennae perform thefunctions of light absorption and excitation energy transfer to aphotochemical reaction center (see, e.g., Simpson and Knoetzel, In: OrtD R and Yocum C F (eds), Oxygenic Photosynthesis: The Light Reactions,pp. 493-506, Kluwer Academic Publishers, Dordrecht, The Netherlands,1996; Pichersky and Jansson, In: Ort D R and Yocum C F (eds), OxygenicPhotosynthesis: The Light Reactions, pp. 507-521, Kluwer AcademicPublishers, Dordrecht, The Netherlands, 1996). Up to 350 chlorophyll a(Chl a) and Chl b molecules can be found in association with PSII,whereas the Chl antenna size of PSI may contain up to 300 mainly Chl amolecules (Melis, Biochim. Biophys. Acta (Reviews on Bioenergetics)1058: 87-106, 1991; Melis, In, Oxygenic Photosynthesis: The LightReactions” (D R Ort, C F Yocum, eds), Kluwer Academic Publishers,Dordrecht, The Netherlands, pp. 523-538, 1996). Some of these Chlmolecules are contained within the PS-core complexes, which are highlyconserved in all organisms of oxygenic photosynthesis. The PSII-corecomplex contains about 37 Chl a molecules, whereas the PSI-core complexcontains 95 Chl a molecules (Glick & Melis, Biochim Biophys Acta 934:151-155, 1988; Jordan et al., Nature 411(6840): 909-917, 2001; Zouni etal., Nature 409: 739-743, 2001; Ruban et al., Nature 421: 648-652,2003). In green plants and algae, the remaining Chl a and Chl b antennamolecules are organized within 10 peripheral subunits of the so-calledauxiliary chlorophyll a-b light-harvesting complex. There are six suchsubunits for PSII (Lhc b1-b6) and four for PSI (Lhc a1-a4) (Jansson etal., Plant Mol Biol Rep, 10: 242-253, 1992). These peripheral Lhcsubunits are not essential for the process of photosynthesis. Indeed,when the chloroplast development is limited, stable assembly of thePSII-core and PSI-core complexes takes place in the absence of any Lhcproteins (Glick & Melis, 1988, supra).

A genetic tendency of photosynthetic organisms to assemble large arraysof light absorbing Chl antenna molecules in their photosystems is asurvival strategy and a competitive advantage in the wild, where lightis often limiting (Kirk, Light and photosynthesis in aquatic ecosystems,2nd edn. Cambridge University Press, Cambridge, England, 1994). However,the Chl antenna size of the photosystems is not fixed but can varysubstantially depending on developmental, genetic, physiological andeven environmental conditions (Melis, 1991, supra). It is recognized inthe field that a genetic regulatory mechanism dynamically modulates theChl antenna size of photosynthesis (Anderson, Annu Rev Plant Physiol 37:93-136, 1986; Escoubas et al., Proc. Nat. Acad. Sci. 92: 10237-10241,1995; Melis, 1991 and 1996, both supra; Melis, Intl. J. Hydrogen Energy27: 1217-1228, 2002; Melis, Chapter 12 in Artificial Photosynthesis:From Basic Biology to Industrial Application, A F Collins and CCritchley (eds.), Wiley-Verlag & Co., pp. 229-240, 2005). For example,the Chl antenna size is adjusted and optimized in response to the lightintensity during plant growth (Ley and Mauzerall, Biochim Biophys Acta680: 95-106, 1982; Sukenik et al., Biochim Biophys Acta 932: 206-215,1988; Smith et al., Plant Physiol. 93: 1433-1440, 1990; LaRoche et al.,Plant Physiol 97: 147-153, 1991; Maxwell et al., Plant Physiol 107:687-694, 1995; Falbel et al., Plant Physiol. 112: 821-832, 1996; Webband Melis, Plant Physiol. 107: 885-893, 1995; Ohtsuka et al., PlantPhysiol. 113: 137-147, 1997; Tanaka and Melis, Plant Cell Physiol. 38:17-24, 1997; Masuda et al., Plant Physiol. 128: 603-614, 2002).Physiological and biochemical consequences of the function of thismolecular regulatory mechanism for the Chl antenna size are wellunderstood. However, little is known about the genes and proteins andtheir mode of action in this regulation. The Chl antenna size regulatorymechanism is highly conserved and functions in all organisms of oxygenicand anoxygenic photosynthesis (Anderson, Annu Rev Plant Physiol 37:93-136, 1986; Nakada et al., J Ferment Bioengin 80: 53-57, 1995;Escoubas et al., Proc. Nat. Acad. Sci. 92: 10237-10241, 1995; Huner etal., Trends in Plant Science, 3: 224-230, 1998; Yakovlev et al., FEBSLett 512: 129-132, 2002; Masuda et al., Plant Physiol. 128: 603-614,2002; Masuda et al., Plant Mol. Biol. 51: 757-771, 2003). Thus,identification of the relevant genes and elucidation of the geneticmechanism for the regulation of the Chl antenna size in Chlamydomonasreinhardtii can apply to all photosynthetic organisms.

Although a smaller Chl antenna size may compromise the ability of aplant, e.g., algae, to survive in the wild, in a high-densitycultivation environment, a smaller chlorophyll antenna size would helpto diminish the over-absorption and wasteful dissipation of excitationenergy by the first layer of leaves, cells or chloroplasts, and wouldalso help diminish photoinhibition of photosynthesis at the surfacewhile permitting greater transmittance of light deeper into the culture.Such altered optical properties of the cells would result in greaterphotosynthetic productivity and enhanced solar conversion efficiency bythe high-density culture.

Previous work (Masuda et al. 2003, supra; Polle et al., Planta 217:49-59, 2003, Melis, 2005, supra) described the isolation of tla1, aChlamydomonas reinhardtii DNA insertional mutant having a truncatedlight-harvesting chlorophyll antenna size (Polle et al, 2003, supra).Although these studies identified a mutant that had reduced antennasize, there was no teaching of whether the phenotype was associated withincreased or suppressed tla1 expression. Accordingly, there is a needfor further elucidation of mechanism of Tla1-mediated changes inchlorophyll antenna size.

BRIEF SUMMARY OF THE INVENTION

The current invention is based on the discovery that suppression of Tla1expression results in reduced chlorophyll antenna size. Thus, in oneaspect, the invention provides a method of decreasing chlorophyllantenna size in a plant, e.g., green algae, the method comprising:inhibiting expression of a Tla1 nucleic acid in the plant by introducinginto the plant an expression cassette comprising a promoter operablylinked to a polynucleotide, or a complement thereof, that specificallyhybridizes to a nucleic acid that has at least 70% identity, often atleast 80%, 90%, or 95% identity, to at least 200 contiguous nucleotidesof a sequence encoding SEQ ID NO:2; and selecting a plant with decreasedchlorophyll antenna size compared to a plant in which the expressioncassette has not been introduced. The promoter may be inducible orconstitutive. In some embodiments the polynucleotide is operably linkedto the promoter in the antisense orientation; in other embodiments, thepolynucleotide is operably linked to the promoter in the senseorientation.

In some embodiment, the polynucleotide introduced into the plant, e.g.,green algae, is an siRNA. In other embodiments, the polynucleotide is anantisense RNA.

The nucleic acid to which the polynucleotide hybridizes can encode apolypeptide of SEQ ID NO:2. In particular embodiments, the nucleic acidis SEQ ID NO:3 or SEQ ID NO:1.

Often the plant, e.g., green algae, into which the nucleic acid isintroduced, is selected from Chlamydomonas reinhardtii, Scenedesmusobliquus, Chlorella vulgaris, Botryococcus braunii, Botryococcussudeticus, Dunaliella salina, or Haematococcus pluvialis.

The invention also provides a plant comprising an expression cassettecomprising a polynucleotide, or a complement thereof, that specificallyhybridizes to a nucleic acid that has at least 70% percent identity,often at least 80%, 90%, or 95% identity, to at least 200 contiguousnucleotides of a sequence encoding SEQ ID NO:2. In preferredembodiments, the plant is a green algae, e.g., Chlamydomonasreinhardtii, Scenedesmus obliquus, Chlorella vulgaris, Botryococcusbraunii, Botryococcus sudeticus, Dunaliella salina, or Haematococcuspluvialis.

The invention additionally provides a method of enhancing yields ofphotosynthetic productivity under high-density growth conditions, themethod comprising cultivating a Tla1-suppressed plant of the invention,e.g., green algae such as Chlamydomonas reinhardtii, Scenedesmusobliquus, Chlorella vulgaris, Botryococcus braunii, Botryococcussudeticus, Dunaliella salina, or Haematococcus pluvialis, under brightsunlight and high density growth conditions.

Additionally, the invention provides a method of enhancing H₂production, the method comprising suppressing Tla1 gene expression in agreen algae, e.g., Chlamydomonas reinhardtii, Scenedesmus obliquus, orChlorella vulgaris, to be used for H₂ production; and cultivating thealgae under conditions in which H₂ is produced.

The invention further provides a method of enhancing bio-oil orbio-diesel production, the method comprising suppressing Tla1 geneexpression in a green algae, e.g., Botryococcus braunii or Botryococcussudeticus to be used for bio-oil or bio-diesel production; andcultivating the algae under conditions in which bio-oil or bio-diesel isproduced.

Further, the invention provides a method of enhancing beta-carotene,lutein or zeaxanthin production, the method comprising suppressing Tla1gene expression in a green algae, e.g., Dunaliella salina, to be usedfor beta-carotene, lutein or zeaxanthin production; and cultivating thealgae under conditions in which beta-carotene, lutein or zeaxanthin isproduced.

In other embodiments, the invention provides a method of enhancingastaxanthin production, the method comprising suppressing Tla1 geneexpression in a green algae, e.g., Haematococcus pluvialis, to be usedfor astaxanthin production; and cultivating the algae under conditionsin which astaxanthin is produced.

In another aspect, the invention provides a method of screening forplants, preferably, green algae, that show enhanced yield ofphotosynthetic productivity, the method comprising: introducing amutation into a population of plants, e.g., green algae; and screeningfor inhibition of Tla1 gene expression, wherein inhibition of Tla1 geneexpression is determined by measuring the level of Tla1 mRNA or Tla1protein. Preferably the plants, e.g., green algae, are selected fromChlamydomonas reinhardtii, Scenedesmus obliquus, Chlorella vulgaris,Botryococcus braunii, Botryococcus sudeticus, Dunaliella salina, orHaematococcus pluvialis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A) Map of plasmid pJD67 insertion in the tla1 mutant genomicDNA. There is a single plasmid insert, containing the ARG7.8 gene. Anapproximately 2.3 kb segment of the 5′ end and 9 base pairs at the 3′end of the pJD67 were deleted upon plasmid insertion. About 6 kb of C.reinhardtii genomic DNA in the tla1 mutant was also deleted from thesite of plasmid insertion. Probes for screening tla1 partial genomiclibraries to clone 5′- and 3′-insert flanking regions are shown by theSalI-SalI and NdeI-NdeI restriction sites on the map. (B) Gene structureof the tla1 mutant and wild type C. reinhardtii in the pJD67 plasmidinsertion locus: 104 bp of 5′ UTR, a total coding region of 642 bp(coding region of exon 1 with 198 bp and coding region of exon 2 with444 bp), a single intron of 116 bases and 1.26 kb of 3′ UTR, encoding aprotein of 213 amino acids.

FIG. 2. Genomic DNA map showing the Tla1 gene structure in: (A) the hostCC425 strain, (B) the tla1 mutant, and (C) the Tla1 complementingplasmid containing the ble gene. Note that the tla1-complements willhave both the mutant gene shown in (B) and the wild type gene shown in(C). Dotted rectangles denote the promoter region of the Tla1 gene;Small hatched rectangles denote the 5′UTR of the Tla1 gene; Long-hatchedrectangles denote the 3′UTR of the Tla1 gene. Thick black arrows andblack lines denote the Tla1 exons and introns, respectively. Primers 1,2, 3, 4, 5, 6, 7, 8 and 9 were used for PCR analysis and are denoted bysmall black arrows on the genomic DNA map (see Table 1).

FIG. 3. PCR analysis of wild type and tla1 mutant. The presence oftranscripts of the Tla1 gene was tested by RT-PCR with primers fromdifferent regions of the Tla1 cDNA. Total RNA was isolated fromTBP-grown Chlamydomonas reinhardtii wild type and tla1 mutant cultures.The down stream PCR primer, “primer 5” was designed from the exon 2region of the Tla1 gene and were the same for all lanes in thisexperiment. Lanes 1, 2: upstream primer “primer 2” was designed from the5′ UTR region (“primer 2”—GCCTGCCACAACCTCAGACCAAGAGACG (SEQ ID NO:15));expected product size of 454 bp). Lanes 4-6: upstream primers weredesigned from the exon 1 region of the Tla1 gene (“primer3”—GGGCCCTTCAGCTGCTCCGCTGACCAAACC (SEQ ID NO:10)). Lanes 4, 5: expectedproduct size of 409 bp., Lane 6: genomic DNA was isolated from the tla1mutant and used as a template for the PCR reaction; expected productsize of 525 bp, i.e., larger than those of lanes 4, 5, due to thepresence of a 116 bp intron, existing between exons 1 and 2. The 1.5%agarose gel was also loaded with M markers (Lane 3) containing a 1 kbDNA ladder (Promega, Madison, Wis.). The PCR products in lane 4 and 5aligned at the 396 bp marker. The PCR products in lane 6 aligned at aposition slightly higher than the 506-517 bp markers.

FIG. 4. 5′ RACE DNA sequence analysis of wild type (cw15) and tla1mutant and sequence comparison with the 3′ end of pJD67. Upper panel:DNA sequence obtained from the 5′ RACE of the wild type (SEQ ID NO:16).Unshaded nucleotides represent the 5′ UTR of the cDNA sequence amplifiedfrom the WT-Tla1 gene transcripts. The ATG start codon is denoted inbold characters. Exon 1 nucleotides are shown in shaded upper casecharacters. Middle panel: DNA sequence obtained from the 5′ RACE of thetla1 mutant (SEQ ID NO:17). The underlined lower case letters representthe apparent 5′ UTR sequence amplified from the tla1 mutant. Exon 1nucleotide sequences are shown in shaded upper case characters. Lowerpanel: 3′ end DNA sequence of plasmid pJD67 (SEQ ID NO:18). Shaded uppercase characters correspond to the DNA sequence of the ARG7.8 gene,whereas lower case characters correspond to the 3′ end of the vectorsequence. Rearrangements in that portion of the plasmid are shown inbold characters, as follows: the last 9 plasmid bases “a t t a a a g ct” were deleted during the plasmid insertion.

FIG. 5. Mapping of BAC clone 39e16 and subcloning of full length Tla1gene for tla1 mutant complementation experiments. Southern blot analysisof the DNA from BAC clone 39e16 and subsequent DNA sequencing ofsubclones provided information on size and locus of a 3.7 kb Apa I-Apa IDNA fragment and a 3 kb Pst I-Pst I fragment. The 2 kb overlappingsegment of the Pst I-Pst I fragment was removed. The remainder Pst I-PstI piece was ligated onto the Apa I-Apa I DNA fragment and cloned inpBluescript to yield the 4.7 kb full length Tla1 gene on a singleplasmid for use in complementation experiments.

FIG. 6. TAP-Agar plate showing wild type (WT), tla1 mutant andcomplemented strains of the latter. Mutant strains were complementedwith a copy of the wild type Tla1 gene. The phenotype of the tla1 mutantshowed a faint green coloration, indicative of the low-level chlorophyllconcentration in the cells, whereas the WT and putative complements 1, 2and 3 were of about the same dark green coloration, indicating a greaterChl/cell.

FIG. 7. PCR analysis using genomic DNA of wild type and tla1complements. (A) PCR product of 593 bp obtained with “primer 1” and“primer 4” (Tla1 promoter/Exon-2) primers. (B) PCR product of 684 bpobtained with “primer 7” and “primer 4” (pJD67 3′end/Tla1 Exon-2)primers. (C) PCR product of 436 bp obtained with “primer 8” and “primer9” (Ble gene) primers. (D) PCR product of 939 bp obtained with “primer1” and “primer 6” (Tla1 promoter/3′UTR) primers. (E) RT-PCR analysis oftla1 complements. PCR product of 823 bp obtained when “primer 1” and“primer 6” (Tla1 5′UTR/3′UTR) specific primers were used. “0”, “W”, “T”,“1”, “2” and “S” stand for zero DNA, wild type, tla1 mutant, tla1-comp1,tla1-comp2 and pSP124s plasmid containing the Ble gene, respectively.

FIG. 8. Overexpression and purification of recombinant Tla1 protein. A12.5% SDS-PAGE gel stained with Coomassie blue showing: (A) Un-induced(lane 1) and induced (lane 2) E. coli cells expressing the recombinant6*His-Tla1 protein. 20 μg of total E. coli cell protein extracts wereloaded on each lane. “M” denotes unstained Benchmark low molecularweight markers. (B) Purified 6*His-Tla1 protein fractions (lanes 1 and2). 35 μg of purified recombinant protein was loaded in each lane. “M”denotes the Benchmark pre-stained low molecular weight markers

FIG. 9. Immune serum titer and Tla1 protein immuno-detection in wildtype and tla1 mutant. (A) A Western blot of isolated recombinant Tla1protein (6*His-Tla1), probed with Tla1-specific antibodies. Lanes 1, 2and 3 contain 20 ng, 20 pg and 2 pg of purified recombinant Tla1protein, respectively. (B) SDS-PAGE stained with Coomassie blue showingthe total protein profile of wild type (W) and tla1 mutant (T) of C.reinhardtii. Lanes were loaded on an equal-Chl basis (6 nmol Chl perlane). “M” stands for the Benchmark pre-stained low molecular weightmarkers. (C) Western blot analysis of wild type (W) and tla1 (T) totalcell protein extracts from C. reinhardtii, probed with Tla1-specificpolyclonal antibodies. Lanes were loaded on an equal-Chl basis (6 nmolChl per lane).

FIG. 10. SDS-PAGE and Western blot analysis of wild type, tla1 mutantand tla1 complemented strains. (A) SDS-PAGE of C. reinhardtii total cellprotein extracts from wild type (W), tla1 mutant (T), and tla1complements comp1 (lane 1) and comp2 (lane 2). Lanes were loaded on anequal-Chl basis (4 nmol Chl per lane). “M” stands for the unstainedBenchmark low molecular weight markers. (B) Western blot analysis of C.reinhardtii total cell protein extracts from wild type (W), tla1 mutant(T), and tla1 complements, tla1-comp1 (lane 1) and tla1-comp2 (lane 2),probed with Tla1-specific polyclonal antibodies.

FIG. 11. Hydropathy plot of the Tla1 deduced amino acid sequence. TheX-axis plots the 213 amino acids of the Tla1 protein, whereas the Y-axisplots the respective amino acid hydropathy index. Positive hydropathyindex corresponds to hydrophobic domains of the protein whereas anegative hydropathy index corresponds to hydrophilic polypeptidedomains.

FIG. 12. (A) Alignment of Tla1-like proteins from different organisms.The alignment of the Tla1 deduced amino acid sequence of C. reinhardtiiis compared to that of similar proteins from A. thaliana (SEQ ID NO:19),O. sativa (SEQ ID NO:20), H. sapiens CGI 112 protein (SEQ ID NO:21), andD. melanogaster (SEQ ID NO:22). Four polypeptide domains with highsequence conservation can be deduced from this comparison. The alignmentwas done on the basis of the ClustalW web-based software(http://www.ch.embnet.org/software/ClustalW.html). (B) Phylogeneticcomparison of putative Tla1 homologue proteins encoded by genes from avariety of organisms. The phylogenetic tree of the above-shown proteinswas based on the deduced amino acid sequences((http://www.ebi.ac.uk/clustalw).

FIG. 13 shows an alignment of Tla1 protein sequences (SEQ ID NOs:20, 23,19 and 2) in plants and algae. Conserved domains in C. reinhardtii Tla1protein=SEQ ID NOs:24-28.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “nucleic acid” and “polynucleotide” are used synonymously andrefer to a single or double-stranded polymer of deoxyribonucleotide orribonucleotide bases read from the 5′ to the 3′ end. A nucleic acid ofthe present invention will generally contain phosphodiester bonds,although in some cases, nucleic acid analogs may be used that may havealternate backbones, comprising, e.g., phosphoramidate,phosphorothioate, phosphorodithioate, or O-methylphophoroamiditelinkages (see Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press); and peptide nucleic acid backbonesand linkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, and non-ribose backbones. Thus, nucleicacids or polynucleotides may also include modified nucleotides, thatpermit correct read through by a polymerase. “Polynucleotide sequence”or “nucleic acid sequence” may include both the sense and antisensestrands of a nucleic acid as either individual single strands or in aduplex. As will be appreciated by those in the art, the depiction of asingle strand also defines the sequence of the complementary strand;thus the sequences described herein also provide the complement of thesequence. Unless otherwise indicated, a particular nucleic acid sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. The nucleic acid may be DNA,both genomic and cDNA, RNA or a hybrid, where the nucleic acid maycontain combinations of deoxyribo- and ribo-nucleotides, andcombinations of bases, including uracil, adenine, thymine, cytosine,guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc

The phrase “nucleic acid sequence encoding” refers to a nucleic acidthat codes for an amino acid sequence of at least 5 contiguous aminoacids within one reading frame. The amino acid need not necessarily beexpressed when introduced into a cell or other expression system, butmay merely be determinable based on the genetic code. For example, thesequence ATGATGGAGCATCAT (SEQ ID NO:29) encodes MMEHH (SEQ ID NO:30).Thus, a polynucleotide may encode a polypeptide sequence that comprisesa stop codon or contains a changed frame so long as at least 5contiguous amino acids within one reading frame. The nucleic acidsequences may include both the DNA strand sequence that is transcribedinto RNA and the RNA sequence. The nucleic acid sequences include boththe full length nucleic acid sequences as well as fragments from thefull length sequences. It should be further understood that the sequenceincludes the degenerate codons of the native sequence or sequences whichmay be introduced to provide codon preference in a specific host cell.

The term “promoter” or “regulatory element” refers to a region orsequence determinants located upstream or downstream from the start oftranscription that are involved in recognition and binding of RNApolymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Such promoters need not be of plant origin, for example,promoters derived from plant viruses, such as the CaMV35S promoter, canbe used in the present invention.

As used herein, the term “algal regulatory element” or “algae promoter”refers to a nucleotide sequence that, when operatively linked to anucleic acid molecule, confers e expression upon the operatively linkednucleic acid molecule in unicellular green algae. It is understood thatlimited modifications can be made without destroying the biologicalfunction of a regulatory element and that such limited modifications canresult in algal regulatory elements that have substantially equivalentor enhanced function as compared to a wild type algal regulatoryelement. These modifications can be deliberate, as through site-directedmutagenesis, or can be accidental such as through mutation in hostsharboring the regulatory element. All such modified nucleotide sequencesare included in the definition of an algal regulatory element as long asthe ability to confer expression in unicellular green algae issubstantially retained.

The term “suppressed” or “decreased” encompasses the absence of Tla1protein in a plant, e.g., algae, as well as protein expression that ispresent but reduced as compared to the level of Tla1 protein expressionin a wild type plant, e.g., algae. The term “suppressed” alsoencompasses an amount of Tla1 protein that is equivalent to wild typelevels, but where the protein has a reduced level of activity incomparison to wild type plants. Generally, at least a 20% decrease inTla1 activity, amount, chlorophyll antenna size or the like ispreferred, with at least about 50% or at least about 75% beingparticularly preferred.

A polynucleotide sequence is “heterologous to” a second polynucleotidesequence if it originates from a foreign species, or, if from the samespecies, is modified by human action from its original form. Forexample, a promoter operably linked to a heterologous coding sequencerefers to a coding sequence from a species different from that fromwhich the promoter was derived, or, if from the same species, a codingsequence which is different from any naturally occurring allelicvariants.

A “Tla1 polynucleotide” is a nucleic acid sequence substantially similarto SEQ ID NO:1 or SEQ ID NO:3, or that encodes a polypeptide that issubstantially similar to SEQ ID NO:2. Tla1 polynucleotides may comprise(or consist of) a region of about 15 to about 3,000 or more nucleotides,sometimes from about 20, or about 50, to about 2,000 nucleotides andsometimes from about 200 to about 600 nucleotides, which hybridizes toSEQ ID NO:1 or SEQ ID NO:3, or the complements thereof, under stringentconditions, or which encodes a Tla1 polypeptide or fragment of at least15 amino acids thereof. Tla1 polynucleotides can also be identified bytheir ability to hybridize under low stringency conditions (e.g., Tm˜40°C.) to nucleic acid probes having the sequence of SEQ ID NO:1 or SEQ IDNO:3. Such Tla1 nucleic acid sequence can have, e.g., about 25-30% basepair mismatches or less relative to the selected nucleic acid probe. SEQID NOs:1 and 3 are exemplary Tla1 polynucleotide sequences. The term“Tla1 polynucleotide” encompasses antisense as well as sense nucleicacids.

A “Tla1 polypeptide” is an amino acid sequence that is substantiallysimilar to SEQ ID NO:2, or a fragment or domain thereof. A full-lengthTla1 protein is 213 amino acids. The majority of the amino acid residuesare hydrophilic, suggesting that it is a soluble cytosolic protein. Asingle hydrophobic domain is present. The domain comprises 27 aminoacids between residues 42 and 69 (with reference to SEQ ID NO:2). Thehydrophobic domain is highly conserved in diverse organisms.

As used herein, a homolog or ortholog of a particular Tla1 gene (e.g.,SEQ ID NO:1) is a second gene in the same plant type or in a differentplant type, which has a polynucleotide sequence of at least 50contiguous nucleotides which are substantially identical (determined asdescribed below) to a sequence in the first gene. It is believed that,in general, homologs or orthologs share a common evolutionary past.

An “expression cassette” refers to a nucleic acid construct, which whenintroduced into a host cell, results in transcription and/or translationof a RNA or polypeptide, respectively. Antisense constructs or senseconstructs that are not or cannot be translated are expressly includedby this definition.

In the case of both expression of transgenes and inhibition ofendogenous genes (e.g., by antisense, or sense suppression) one of skillwill recognize that the inserted polynucleotide sequence need not beidentical and may be “substantially identical” to a sequence of the genefrom which it was derived. As explained below, these variants arespecifically covered by this term.

In the case where the inserted polynucleotide sequence is transcribedand translated to produce a functional polypeptide, one of skill willrecognize that because of codon degeneracy a number of polynucleotidesequences will encode the same polypeptide. These variants arespecifically covered by the term “polynucleotide sequence from” a Tla1gene. In addition, the term specifically includes sequences (e.g., fulllength sequences) substantially identical (determined as describedbelow) with a Tla1 gene sequence. A “polynucleotide sequence from” aTla1 gene can encode a protein that retains the function of a Tla1polypeptide in contributing to chlorophyll antenna size.

In the case of polynucleotides used to inhibit expression of anendogenous gene, the introduced sequence need not be perfectly identicalto a sequence of the target endogenous gene. The introducedpolynucleotide sequence will typically be at least substantiallyidentical (as determined below) to the target endogenous sequence. Thus,an introduced “polynucleotide sequence from” a Tla1 gene may not beidentical to the target Tla1 gene to be suppressed, but is functional inthat it is capable of inhibiting expression of the target Tla1 gene.

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The term “complementary to” is used herein to mean thatthe sequence is complementary to all or a portion of a referencepolynucleotide sequence.

Optimal alignment of sequences for comparison may be conducted by thelocal homology algorithm of Smith and Waterman Add. APL. Math. 2:482(1981), by the homology alignment algorithm of Needle man and Wunsch J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearsonand Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, BLAST,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The term “substantial identity” in the context of polynucleotidesequences means that a polynucleotide comprises a sequence that has atleast 50% sequence identity. Alternatively, percent identity can be anyinteger from 40% to 100%. Exemplary embodiments include at least: 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. compared to areference sequence using the programs described herein; preferably BLASTusing standard parameters, as described below. Accordingly, Tla1sequences of the invention include nucleic acid sequences that havesubstantial identity to SEQ ID NO:1, or a portion of SEQ ID NO:1 such asthe coding region of SEQ ID NO:1, or SEQ ID NO:3.

Tla1 polypeptide sequences of the invention include polypeptidesequences having substantial identify to SEQ ID NO:2. One of skill willrecognize that these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like. Substantial identity of amino acidsequences for these purposes normally means sequence identity of atleast 50%. Preferred percent identity of polypeptides can be any integerfrom 50% to 100%, e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99%, an sometimes at least 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% and 75%. Polypeptides which are“substantially similar” share sequences as noted above except thatresidue positions which are not identical may differ by conservativeamino acid changes. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other, or a third nucleic acid,under stringent conditions. Stringent conditions are sequence dependentand will be different in different circumstances. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.The Tm is the temperature (under defined ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe.Typically, stringent conditions will be those in which the saltconcentration is about 0.02 molar at pH 7 and the temperature is atleast about 60° C.

In the present invention, mRNA encoded by Tla1 genes of the inventioncan be identified in Northern blots under stringent conditions usingcDNAs of the invention or fragments of at least about 100 nucleotides.For the purposes of this disclosure, stringent conditions for suchRNA-DNA hybridizations are those which include at least one wash in0.2×SSC at 63° C. for 20 minutes, or equivalent conditions. Genomic DNAor cDNA comprising genes of the invention can be identified using thesame cDNAs (or fragments of at least about 100 nucleotides) understringent conditions, which for purposes of this disclosure, include atleast one wash (usually 2) in 0.2×SSC at a temperature of at least about50° C., usually about 55° C., for 20 minutes, or equivalent conditions.

A Tla1 gene for use in the invention can also be amplified using PCRtechniques. For example, a Tla1 gene of the invention may be amplifiableby the primer set: (5′ TACGGGAATTTGCGGAACCTC 3′ (SEQ ID NO:4)) and” (5′AACACACACCCCGCACT 3′ (SEQ ID NO:7)).

The term “isolated”, when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state and may be in either a dry or aqueoussolution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest.

INTRODUCTION

The present invention provides methods of suppressing Tla1 geneexpression in plants, e.g., green algae. Plants having suppressed Tla1gene expression exhibit decreases in the size of chlorophyll antenna.Such plants are useful for many purposes. For example, Tla1 suppressioncan be used to enhance plant growth and photosynthetic productivity. Inembodiments where the plant is a green algae, such Tla1-suppressedplants can be used, e.g., in mass culture for production of variousnutrients or pharmaceuticals, for production of H₂, for production oflipid/hydrocarbons, for carbon sequestration, for waste-water treatmentand aquatic pollution amelioration, for flu gas treatment andatmospheric pollution amelioration, for biomass generation, and forother purposes.

A Tla1 nucleic acid that is targeted for suppression in this inventionencodes a Tla1 protein that is substantially similar to SEQ ID NO:2, ora fragment thereof. For example, such Tla1 proteins have one or moreconserved domains, designated with reference to SEQ ID NO2: amino acidpositions 9-33, amino acid positions 41-70, amino acid positions 75-129,amino acid positions 135-163, or amino acid positions 177-200. Otherexemplary plant Tla1-related polynucleotide sequences are from Oryzasativa (Accession No. CX102072), Zea mays (Accession No. EB673149), andArabidopsis thaliana (Accession No. DR308999). Examples of conservedregions of these proteins are shown in FIG. 13. 1. Other exemplaryTla1-related sequences include those from Solanum tuberosum (potato)(Accession No. CV500710); Gossypium arboreum (Accession No. BG44500);Helianthus annuus (Accession No. BQ967999); Nicotiana tabacum (tobacco)(Accession No. EB678062); Triticum aestivum (wheat) (Accession No.CV065526); Hordeum vulgare (barley) (Accession No. AL504185); andGlycine max (soybean) (Accession No. BM107844).

The invention employs various routine recombinant nucleic acidtechniques. Generally, the nomenclature and the laboratory procedures inrecombinant DNA technology described below are those well known andcommonly employed in the art. Many manuals that provide direction forperforming recombinant DNA manipulations are available, e.g., Sambrook &Russell, Molecular Cloning, A Laboratory Manual (3rd Ed, 2001); andCurrent Protocols in Molecular Biology (Ausubel et al., eds.,1994-1999).

TLA1 Nucleic Acid Sequences

Isolation or generation of Tla1 polynucleotide sequence can beaccomplished by a number of techniques. For instance, oligonucleotideprobes based on the sequences disclosed here can be used to identify thedesired polynucleotide in a cDNA or genomic DNA library from a desiredplant species. Such a cDNA or genomic library can then be screened usinga probe based upon the sequence of a cloned Tla1 gene, e.g., SEQ ID NO:1or 3. Probes may be used to hybridize with genomic DNA or cDNA sequencesto isolate homologous genes in the same or different plant species.

Alternatively, the nucleic acids of interest can be amplified fromnucleic acid samples using amplification techniques. For instance, PCRmay be used to amplify the sequences of the genes directly from mRNA,from cDNA, from genomic libraries or cDNA libraries. PCR and other invitro amplification methods may also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of the desiredmRNA in samples, for nucleic acid sequencing, or for other purposes.

Appropriate primers and probes for identifying a Tla1 gene from plantcells, e.g., algae, can be generated from comparisons of the sequencesprovided herein. For a general overview of PCR see PCR Protocols: AGuide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J.and White, T., eds.), Academic Press, San Diego (1990). Exemplary primerpairs are: “primer 1” (5′ TACGGGAATTTGCGGAACCTC 3′ (SEQ ID NO:4)) and“primer 6” (5′ AACACACACCCCGCACT 3′ (SEQ ID NO:7)) set out in Table 1hereinbelow. Exemplary amplification reaction conditions are: 20 mM TrisHCl, pH 8.4, 50 mM potassium chloride, 2.5 mM magnesium chloride, 0.25mM dATP, 0.25 mM dCTP, 0.25 mM dGTP, 0.25 mM dTTP, 0.6 μM primers, and2.5 units Taq polymerase/PCR reaction. An exemplary thermal cyclingprogram is 94° C. for 3 min., 35 cycles of 95° C. for 45 sec, 55° C.-59°C. for 30 sec, 72° C. for 130 sec, followed by 72° C. for 10 min.

The genus of Tla1 nucleic acid sequences for use in the inventionincludes genes and gene products identified and characterized bytechniques such as hybridization and/or sequence analysis usingexemplary nucleic acid sequences, e.g., SEQ ID NOs:1 and 3, and proteinsequences, e.g., SEQ ID NO:2.

Preparation of Recombinant Vectors

To use isolated sequences in the above techniques, recombinant DNAvectors suitable for transformation of plant cells, e.g., green algaecells, are prepared. Techniques for transforming a wide variety ofhigher plant species are well known and described in the technical andscientific literature. See, for example, Weising et al. Ann. Rev. Genet.22:421-477 (1988). For example, a DNA sequence encoding a sequence tosuppress Tla1 expression (described in further detail below), willpreferably be combined with transcriptional and other regulatorysequences which will direct the transcription of the sequence from thegene in the intended cells of the transformed plant.

Regulatory sequences include promoters, which may be either constitutiveor inducible, or where a higher plant is involved, tissue-specific. Forexample, a plant promoter fragment may be employed that is constitutive,i.e., it will direct expression of the gene under most environmentalconditions and states of cell differentiation. Examples of constitutivepromoters include the cauliflower mosaic virus (CaMV) 35S transcriptioninitiation region, the 1′- or 2′-promoter derived from T-DNA ofAgrobacterium tumafaciens, the CaMV 19S promoter; the Figwort mosaicvirus promoter; actin promoters, and the nopaline synthase (nos) genepromoter. Other constitutive promoter include promoters such as theArabidopsis actin gene promoter (see, e.g., Huang (1997) Plant Mol.Biol. 1997 33:125 139); alcohol dehydrogenase (Adh) gene promoters (see,e.g., Millar (1996) Plant Mol. Biol. 31:897 904); ACT11 from Arabidopsis(Huang et al. Plant Mol. Biol. 33:125-139 (1996)), Cat3 promoter fromArabidopsis (Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)), thepromoter from the gene encoding stearoyl-acyl carrier protein desaturasefrom Brassica napus (Solocombe et al. Plant Physiol. 104:1167-1176(1994)), GPc1 promoter from maize (Martinez et al. J. Mol. Biol208:551-565 (1989)), Gpc2 promoter from maize (Manjunath et al., PlantMol. Biol. 33:97-112 (1997)), and other transcription initiation regionsfrom various plant genes known to those of skill. Chimeric regulatoryelements, which combine elements from different genes, also can beuseful for e expressing a nucleic acid molecule encoding a Tlapolynucleotide.

Alternatively, a plant promoter can be used to direct expression of Tla1nucleic acid under the influence of changing environmental conditions.Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions, elevated temperature,or the presence of light. Plant promoters that are inducible uponexposure to chemicals reagents, such as herbicides or antibiotics, arealso used to express Tla1 nucleic acids. For example, the maize In2 2promoter, activated by benzenesulfonamide herbicide safeners, can beused (De Veylder (1997) Plant Cell Physiol. 38:568 577). Other usefulinducible regulatory elements include copper-inducible regulatoryelements (Mett et al., Proc. Natl. Acad. Sci. USA 90:4567-4571 (1993);Furst et al., Cell 55:705-717 (1988)); tetracycline andchlor-tetracycline-inducible regulatory elements (Gatz et al., Plant J.2:397-404 (1992); Röder et al., Mol. Gen. Genet. 243:32-38 (1994); Gatz,Meth. Cell Biol. 50:411-424 (1995)); ecdysone inducible regulatoryelements (Christopherson et al., Proc. Natl. Acad. Sci. USA 89:6314-6318(1992); Kreutzweiser et al., Ecotoxicol. Environ. Safety 28:14-24(1994)); heat shock inducible regulatory elements (Takahashi et al.,Plant Physiol. 99:383-390 (1992); Yabe et al., Plant Cell Physiol.35:1207-1219 (1994); Ueda et al., Mol. Gen. Genet. 250:533-539 (1996));and lac operon elements, which are used in combination with aconstitutively expressed lac repressor to confer, for example,IPTG-inducible expression (Wilde et al., EMBO J. 11:1251-1259 (1992)).An inducible regulatory element also can be, for example, anitrate-inducible promoter, e.g., derived from the spinach nitritereductase gene (Back et al., Plant Mol. Biol. 17:9 (1991)), or alight-inducible promoter, such as that associated with the small subunitof RuBP carboxylase or the LHCP gene families (Feinbaum et al., Mol.Gen. Genet. 226:449 (1991); Lam and Chua, Science 248:471 (1990)), or alight.

In one example, a promoter sequence that is responsive to light may beused to drive expression of a Tla1 nucleic acid construct that isintroduced into Chlamydomonas that is exposed to light (e.g., Hahn, CurrGenet 34:459-66, 1999; Loppes, Plant Mol Biol 45:215-27, 2001; Villand,Biochem J 327:51-7), 1997. Other light-inducible promoter systems mayalso be used, such as the phytochrome/PIF3 system (Shimizu-Sato, NatBiotechnol 20):1041-4, 2002). Further, a promoter can be used that isalso responsive to heat can be employed to drive expression in algaesuch as Chlamydomonas (Muller, Gene 111:165-73, 1992; von Gromoff, MolCell Biol 9:3911-8, 1989). Additional promoters, e.g., for expression inalgae such as green microalgae, include the RbcS2 and PsaD promoters(see, e.g., Stevens et al., Mol. Gen. Genet. 251: 23-30, 1996; Fischer &Rochaix, Mol Genet Genomics 265:888-94, 2001).

In some embodiments, the promoter may be from a gene associated withphotosynthesis in the species to be transformed or another species. Forexample such a promoter from one species may be used to directexpression of a protein in transformed algal cells or cells of anotherphotosynthetic marine organism. Suitable promoters may be isolated fromor synthesized based on known sequences from other photosyntheticorganisms. Preferred promoters are those for genes from otherphotosynthetic species that are homologous to the photosynthetic genesof the algal host to be transformed. For example, a series of lightharvesting promoters from the fucoxanthing chlorophyll binding proteinhave been identified in Phaeodactylum tricornutum (see, e.g., Apt, etal. Mol Gen. Genet. 252:572-579, 1996). In other embodiments, acarotenoid chrlophyll binding protein promoter, such as that ofperidinin chlorophyll binding protein, can be used.

In some embodiments, promoters are identified by analyzing the 5′sequences of a genomic clone corresponding to the Tla1 genes describedhere. Sequences characteristic of promoter sequences can be used toidentify the promoter. Sequences controlling eukaryotic gene expressionhave been extensively studied and include basal elements such as CG-richregions, TATA consensus sequences etc. In plants, further upstream,there is typically a promoter element with a series of adeninessurrounding the trinucleotide G (or T) N G. J. Messing et al., inGENETIC ENGINEERING IN PLANTS, pp. 221-227 (Kosage, Meredith andHollaender, eds. (1983)).

A number of methods are known to those of skill in the art foridentifying and characterizing promoter regions in plant genomic DNA(see, e.g., Jordano, et al., Plant Cell, 1: 855 866 (1989); Bustos, etal., Plant Cell, 1:839 854 (1989); Green, et al., EMBO J. 7, 4035 4044(1988); Meier, et ah, Plant Cell, 3, 309 316 (1991); and Zhang, et al.,Plant Physiology 110: 1069 1079 (1996)). A promoter can be additionallyevaluated by testing the ability of the promoter to drive expression inplant cells, e.g., green algae, in which it is desirable to introduce aTla1 expression construct.

The vector comprising Tla1 nucleic acid sequences will typicallycomprise a marker gene that confers a selectable phenotype on plant oralgae cells. Such markers are known. For example, the marker may encodebiocide resistance, particularly antibiotic resistance, such asresistance to zeocin, kanamycin, G418, bleomycin, hygromycin, orherbicide resistance, such as resistance to chlorosluforon or Basta. Insome embodiments, selectable markers for use in Chlamydomonas can bemarkers that provide spectinomycin resistance (Fargo, Mol Cell Biol19:6980-90, 1999), kanamycin and amikacin resistance (Bateman, Mol-GenGenet 263:404-10, 2000), zeomycin and phleomycin resistance (Stevens,Mol Gen Genet 251:23-30, 1996), and paramomycin and neomycin resistance(Sizova, Gene 277:221-9, 2001).

Tla1 nucleic acid sequences of the invention can be expressedrecombinantly in plant cells, e.g., green algae, or other host cellexpression systems, such as bacteria, yeast, and the like, to increaselevels of Tla1 polypeptides. As appreciated by one of skill in the art,expression constructs can be designed taking into account suchproperties as codon usage frequencies of the organism in which the Tla1nucleic acid is to be expressed. Tla1 polypeptides can be used, e.g.,for the production of antibodies to monitor Tla1 expression.Alternatively, antisense or other Tla1 constructs are used to suppressTla1 levels of expression.

A variety of different expression constructs, such as expressioncassettes and vectors suitable for transformation of plant cells can beprepared. Techniques for transforming a wide variety of plant speciesare well known and described in the technical and scientific literature.See, e.g., Weising et al. Ann. Rev. Genet. 22:421-477 (1988). Forexample, a Tla1 nucleic acid construct can be directly introduced into aplant or algae cell by microparticle bombardment, or using a glass beadmethod (e.g., Kindle, Proc. Natl. Acad. Sci. USA 87: 1228-1232, 1990).Alternatively, e.g., when transfecting higher plants, the DNA constructsmay be combined with suitable T-DNA flanking regions and introduced intoa conventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Other techniques are alsoknown. For example, the introduction of DNA constructs usingpolyethylene glycol precipitation is described in Paszkowski et al. EMBOJ. 3:2717-2722 (1984). Electroporation techniques are described in Frommet al., Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistictransformation techniques are described in Klein et al., Nature327:70-73 (1987).

In some embodiments, Tla1 nucleic acid constructs are introduced intoalgae, e.g., green algae. As noted above, the nuclear, mitochondrial,and chloroplast genomes can be transformed through a variety of knownmethods (see, e.g., Kindle, J Cell Biol 109:2589-601, 1989; Kindle, ProcNatl Acad Sci USA 87:1228-32, 1990; Kindle, Proc Natl Acad Sci USA88:1721-5, 1991; Shimogawara, Genetics 148:1821-8, 1998; Boynton,Science 240:1534-8, 1988; Boynton, Methods Enzymol 264:279-96, 1996;Randolph-Anderson, Mol Gen Genet 236:235-44, 1993).

Suppression of Tla1 Expression

The invention provides methods for generating a plant having a reducedchlorophyll antenna size by suppressing expression of a nucleic acidmolecule encoding Tla1. In a transgenic plant of the invention, anucleic acid molecule, or antisense constructs thereof, encoding a Tla1gene product can be operatively linked to an exogenous regulatoryelement. The invention provides, for example, a transgenic plantcharacterized by reduced chlorophyll antenna size having an expressednucleic acid molecule encoding a Tla1 gene product, or antisenseconstruct thereof, that is operatively linked to an exogenousconstitutive regulatory element. In one embodiment, the inventionprovides a transgenic plant that is characterized by small chlorophyllantenna size due to suppression of a nucleic acid molecule encoding aTla1 polypeptide. Such a plant typically comprises an expressioncassette stably transfected into the plant cell, such that that Tla1polypeptide expression is inhibited constitutively or under certainconditions, e.g., when an inducible promoter is used.

Tla1 nucleic acid sequences can be used to prepare expression cassettesuseful for inhibiting or suppressing Tla1 expression. A number ofmethods can be used to inhibit gene expression in plants. For instance,siRNA, antisense, or ribozyme technology can be conveniently used. Forexample, in Chlamydomonas, antisense inhibition can be used to decreaseexpression of a targeted gene (e.g., Schroda, Plant Cell 11:1165-78,1999). Alternatively, an RNA interference construct can be used (e.g.,Schroda, Curr Genet. 49:69-84, 2006, Epub 2005 Nov. 25).

For antisense expression, a nucleic acid segment from the desired Tla1gene is cloned and operably linked to a promoter such that the antisensestrand of RNA will be transcribed. The expression cassette is thentransformed into plants, e.g., algae, and the antisense strand of RNA isproduced. The antisense nucleic acid sequence transformed into plantswill be substantially identical to at least a portion of the endogenousgene or genes to be repressed. The sequence, however, does not have tobe perfectly identical to inhibit expression. Thus, an antisense orsense nucleic acid molecule encoding only a portion of Tla1 can beuseful for producing a plant in which Tla1 expression is suppressed. Thevectors of the present invention can be designed such that theinhibitory effect applies to other proteins within a family of genesexhibiting homology or substantial homology to the target gene.

For antisense suppression, the introduced sequence also need not be fulllength relative to either the primary transcription product or fullyprocessed mRNA. Generally, higher homology can be used to compensate forthe use of a shorter sequence. Furthermore, the introduced sequence neednot have the same intron or exon pattern, and homology of non-codingsegments may be equally effective. Normally, a sequence of between about30 or 40 nucleotides and about full length nucleotides should be used,though a sequence of at least about 100 nucleotides is preferred, asequence of at least about 200 nucleotides is more preferred, and asequence of at least about 500 nucleotides is especially preferred.SEquences can also be longer, e.g., 1000 or 2000 nucleotides are greaterin length.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of Tla1 genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA cleaving activity upon them,thereby increasing the activity of the constructs.

A number of classes of ribozymes have been identified. One class ofribozymes is derived from a number of small circular RNAs that arecapable of self-cleavage and replication in plants. Ribozymes, e.g.,Group I introns, have also been identified in the chloroplast of greenalgae (see, e.g., Cech, Annu Rev Biochem 59:543-568, 1990; Bhattacharya,Molec Biol and Evol 13: 978-989, 1996; Erin, et al., Amer J Botany90:628-633, 2003; Turmel, et al., Nucl Acids Res. 21:5242-5250, 1993;and Van Oppen et al., Molec Biol and Evol 10:1317-1326, 1993). Thedesign and use of target RNA-specific ribozymes is described, e.g., inHaseloff et al. Nature, 334:585-591 (1988).

Another method of suppression is sense suppression (also known asco-suppression). Introduction of expression cassettes in which a nucleicacid is configured in the sense orientation with respect to the promoterhas been shown to be an effective means by which to block thetranscription of target genes. For an example of the use of this methodto modulate expression of endogenous genes see, Napoli et al., The PlantCell 2:279-289 (1990); Flavell, Proc. Natl. Acad. Sci., USA 91:3490-3496(1994); Kooter and Mol, Current Opin. Biol. 4:166-171 (1993); and U.S.Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.

Generally, where inhibition of expression is desired, some transcriptionof the introduced sequence occurs. The effect may occur where theintroduced sequence contains no coding sequence per se, but only intronor untranslated sequences homologous to sequences present in the primarytranscript of the endogenous sequence. The introduced sequence generallywill be substantially identical to the endogenous sequence intended tobe repressed. This minimal identity will typically be greater than about65%, but a higher identity might exert a more effective repression ofexpression of the endogenous sequences. Substantially greater identityof more than about 80% is preferred, though about 90% or 95% to absoluteidentity would be most preferred. As with antisense regulation, theeffect should apply to any other proteins within a similar family ofgenes exhibiting homology or substantial homology.

For sense suppression, the introduced sequence in the expressioncassette, needing less than absolute identity, also need not be fulllength, relative to either the primary transcription product or fullyprocessed mRNA. This may be preferred to avoid concurrent production ofsome plants that are overexpressers. A higher identity in a shorter thanfull length sequence compensates for a longer, less identical sequence.Furthermore, the introduced sequence need not have the same intron orexon pattern, and identity of non-coding segments will be equallyeffective. Normally, a sequence of the size ranges noted above forantisense regulation is used.

Endogenous gene expression may also be suppressed by means of RNAinterference (RNAi), which uses a double-stranded RNA having a sequenceidentical or similar to the sequence of the target TLA1 gene. RNAi isthe phenomenon in which when a double-stranded RNA having a sequenceidentical or similar to that of the target gene is introduced into acell, the expressions of both the inserted exogenous gene and targetendogenous gene are suppressed. The double-stranded RNA may be formedfrom two separate complementary RNAs or may be a single RNA withinternally complementary sequences that form a double-stranded RNA. Theintroduced double-stranded RNA is initially cleaved into smallfragments, which then serve as indexes of the target gene in somemanner, thereby degrading the target gene. RNAi is known to be alsoeffective in plants (see, e.g., Chuang, C. F. & Meyerowitz, E. M., Proc.Natl. Acad. Sci. USA 97: 4985 (2000); Waterhouse et al., Proc. Natl.Acad. Sci. USA 95:13959-13964 (1998); Tabara et al. Science 282:430-431(1998)). For example, to achieve suppression of the expression of a DNAencoding a protein using RNAi, a double-stranded RNA having the sequenceof a DNA encoding the protein, or a substantially similar sequencethereof (including those engineered not to translate the protein) orfragment thereof, is introduced into a plant of interest, e.g., greenalgae. The resulting plants may then be screened for a phenotypeassociated with the target protein and/or by monitoring steady-state RNAlevels for transcripts encoding the protein. Although the genes used forRNAi need not be completely identical to the target gene, they may be atleast 70%, 80%, 90%, 95% or more identical to the target gene sequence.See, e.g., U.S. Patent Publication No. 2004/0029283. The constructsencoding an RNA molecule with a stem-loop structure that is unrelated tothe target gene and that is positioned distally to a sequence specificfor the gene of interest may also be used to inhibit target geneexpression. See, e.g., U.S. Patent Publication No. 2003/0221211.

The RNAi polynucleotides may encompass the full-length target RNA or maycorrespond to a fragment of the target RNA. In some cases, the fragmentwill have fewer than 100, 200, 300, 400, 500 600, 700, 800, 900 or 1,000nucleotides corresponding to the target sequence. In addition, in someembodiments, these fragments are at least, e.g., 15, 20, 25, 30, 50,100, 150, 200, or more nucleotides in length. In some cases, fragmentsfor use in RNAi will be at least substantially similar to regions of atarget protein that do not occur in other proteins in the organism ormay be selected to have as little similarity to other organismtranscripts as possible, e.g., selected by comparison to sequences inanalyzing publicly-available sequence databases. Thus, RNAi fragmentsmay be selected for similarity or identity with the N terminal region ofthe Tla1 sequences of the invention (i.e., those sequences lackingsignificant homology to sequences in the databases) or may be selectedfor identity or similarity to conserved regions of Tla1 proteins, e.g.,the hydrophobic region.

Expression vectors that continually express siRNA in transiently- andstably-transfected cells have been engineered to express small hairpinRNAs, which get processed in vivo into siRNAs molecules capable ofcarrying out gene-specific silencing (Brummelkamp et al., Science296:550-553 (2002), and Paddison, et al., Genes & Dev. 16:948-958(2002)). Post-transcriptional gene silencing by double-stranded RNA isdiscussed in further detail by Hammond et al. Nature Rev Gen 2: 110-119(2001), Fire et al. Nature 391: 806-811 (1998) and Timmons and FireNature 395: 854 (1998).

One of skill in the art will recognize that using technology based onspecific nucleotide sequences (e.g., antisense or sense suppressiontechnology), families of homologous genes can be suppressed with asingle sense or antisense transcript. For instance, if a sense orantisense transcript is designed to have a sequence that is conservedamong a family of genes, then multiple members of a gene family can besuppressed. Conversely, if the goal is to only suppress one member of ahomologous gene family, then the sense or antisense transcript should betargeted to sequences with the most variation between family members.

Screening for Plants Having Suppressed Tla1 Expression

The invention also provides methods of screening for plants, e.g., greenalgae, having reduced Tla1 gene expression. Such plants can be generatedusing the techniques described above to target Tla1 genes. In otherembodiments mutagenized plants, e.g., algae, can be screened for reducedTla1 gene expression.

Methods for introducing genetic mutations into plant genes and selectingplants with desired traits are well known. For instance, plant cells canbe treated with a mutagenic chemical substance, according to standardtechniques. Such chemical substances include, but are not limited to,the following: diethyl sulfate, ethylene imine, ethyl methanesulfonateand N-nitroso-N-ethylurea. Alternatively, ionizing radiation fromsources such as, X-rays or gamma rays can be used. In other embodiments,insertional mutagenesis can be performed (see, e.g., Polle et al.,Planta 217:49-59, 2003).

Alternatively, homologous recombination can be used to induce targetedgene modifications by specifically targeting a Tla1 gene in vivo tosuppress expression (see, generally, Grewal and Klar, Genetics 146:1221-1238 (1997) and Xu et al., Genes Dev. 10: 2411-2422 (1996)).Homologous recombination has been demonstrated in plants (Puchta et al.,Experientia 50: 277-284 (1994), Swoboda et al., EMBO J. 13: 484-489(1994); Offringa et al., Proc. Natl. Acad. Sci. USA 90: 7346-7350(1993); and Kempin et al. Nature 389:802-803 (1997)).

In applying homologous recombination technology to the genes of theinvention, mutations in selected portions of Tla1 gene sequences(including 5′ upstream, 3′ downstream, and intragenic regions) such asthose disclosed here are made in vitro and then introduced into thedesired plant using standard techniques. Since the efficiency ofhomologous recombination is known to be dependent on the vectors used,use of dicistronic gene targeting vectors as described by Mountford etal., Proc. Natl. Acad. Sci. USA 91: 4303-4307 (1994); and Vaulont etal., Transgenic Res. 4: 247-255 (1995) are conveniently used to increasethe efficiency of selecting for decreased Tla1 gene expression intransgenic plants. The mutated gene will interact with the targetwild-type gene in such a way that homologous recombination and targetedreplacement of the wild-type gene will occur in transgenic plant cells,resulting in suppression of Tla1 activity.

Alternatively, oligonucleotides composed of a contiguous stretch of RNAand DNA residues in a duplex conformation with double hairpin caps onthe ends can be used. The RNA/DNA sequence is designed to align with thesequence of the target Tla1 gene and to contain the desired nucleotidechange. Introduction of the chimeric oligonucleotide on anextrachromosomal T-DNA plasmid results in efficient and specific Tla1gene conversion directed by chimeric molecules in a small number oftransformed plant cells. This method is described in Cole-Strauss etal., Science 273:1386-1389 (1996) and Yoon et al., Proc. Natl. Acad.Sci. USA 93: 2071-2076 (1996).

In other embodiments, insertional mutagenesis can be used to mutagenizea population of plants, e.g., green algae, that can subsequently bescreened.

Plants, e.g., green algae, with mutations can be screened for decreasedTla1 gene expression. Such decreases are determined by examining levelsof Tla1 gene or protein expression. Techniques for performing such ananalysis are readily known in the art and include quantitative RT-PCR,northern blots, immunoassays, and the like. Tla1 expression can also beevaluated by analyzing a phenotypic endpoint such as chlorophyll antennasize and selecting plants having reduce chlorophyll antenna sizerelative to normal.

Plants that can be Targeted

Tla1 can be suppressed in any number of eukaryotic green plants where itis desirable to reduce the rate of light absorption. For example, cropplants, such as tobacco, soybeans, barley, maize, and others (see, e.g.,Okabe, et al., J Plant Physiol. 60: 150-156, 1977; Melis & Thielen,Biochim. Biophys. Acta 589: 275-286, 1980; Ghirardi et al., Biochim.Biophys. Acta 851: 331-339, 1986; Ghirardi & Melis, Biochim. Biophys.Acta 932: 130-137, 1988; Droppa, et al., Biochim. Biophys. Acta 932:138-145, 1988; and Greene, et al., Plant Physiol. 87: 365-370, 1988).

Uses of Tla1 Suppressed Algae

In some embodiments, Tla1 is suppressed in algae. Algae, alga or thelike, refer to plants belonging to the subphylum Algae of the phylumThallophyta. The algae are unicellular, photosynthetic, anoxygenic algaeand are non-parasitic plants without roots, stems or leaves; theycontain chlorophyll and have a great variety in size, from microscopicto large seaweeds. Green algae, which are single cell eukaryoticorganisms of oxygenic photosynthesis endowed with chlorophyll a andchlorophyll b belonging toEukaryota-Viridiplantae-Chlorophyta-Chlorophyceae, are often a preferredtarget. For example, Tla1 expression can be suppressed in C.reinhardtii, which is classified as Volvocales-Chlamydomonadaceae. Algaestrains that are of particular interest for this invention are, e.g.,Chlamydomonas reinhardtii, Scenedesmus obliquus, Chlorella vulgaris,Botryococcus braunii, Botryococcus sudeticus, Dunaliella salina, andHaematococcus pluvialis.

Algae can be used in high density photobioreactors (see, e.g., Lee etal., Biotech. Bioengineering 44:1161-1167, 1994; Chaumont, J Appl.Phycology 5:593-604, 1990), bioreactors for sewage and waste watertreatments (e.g., Sawayama et al., Appl. Micro. Biotech., 41:729-731,1994; Lincoln, Bulletin De L'institut Oceangraphique (Monaco),12:109-115, 1993), elimination of heavy metals from contaminated water(e.g., Wilkinson, Biotech. Letters, 11:861-864, 1989), the production ofβ-carotene (e.g., Yamaoka, Seibutsu-Kogaku Kaishi, 72:111-114, 1994),the production of hydrogen (e.g., U.S. Patent Application PublicationNo. 20030162273), and pharmaceutical compounds (e.g., Cannell, 1990), aswell as nutritional supplements for both humans and animals (Becker,1993, “Bulletin De L'institut Oceanographique (Monaco), 12, 141-155) andfor the production of other compounds of nutritional value.

Conditions for growing Tla1-suppressed algae for the exemplary purposesillustrated above are known in the art (see, e.g., the exemplaryreferences cited herein).

EXAMPLES Methodology Growth of the Algae

Chlamydomonas reinhardtii strain cw15, the arginine-requiring CC425, thechlorophyll-deficient mutant tla1 and Tla1-complemented strains of thetla1 mutant were grown to the mid-exponential growth phase either in TAP[Tris Acetate Phosphate, pH 7.4], TAP+Arg (Sueoka, Proc. Natl. Acad.Sci. USA 46: 83-91, 1960; Harris, The Chlamydomonas source book: Acomprehensive guide to biology and laboratory use: Academic Press, SanDiego, 1989), or in modified minimal media containing 40 mM Tris-HCl, pH7.4, supplemented with 25 mM sodium bicarbonate with or without Arg (TBPmedium, Polle et al., Planta 211: 335-344, 2000) in flat 1-1 Rouxbottles at 25° C. under continuous illumination of 200 μmol photons m-2s-1 provided by cool-white fluorescent lamps. The cultures were stirredcontinuously to ensure a uniform illumination of the cells and toprevent settling.

Cell Count and Chlorophyll Determinations

Cell density was estimated upon counting the number of cells per mlculture using a Neubauer ultraplane hemacytometer. Pigments from intactcells were extracted in 80% acetone and cell debris removed bycentrifugation at 10,000 g for 5 min. The absorbance of the supernatantwas measured with a Shimadzu UV-160U spectrophotometer and thechlorophyll (a and b) concentration of the samples was determinedaccording to Arnon, Plant Physiol 24: 1-15, 1949, with equationscorrected as in Melis et al. (Photochem. Photobiol. 45: 129-136, 1987).

Nucleic Acid Extractions

Genomic DNA was isolated using either Stratagene's (La Jolla, Calif.)DNA purification kit or a combination of QIAGEN's (Valencia, Calif.)DNeasy plant mini kit and phenol chloroform extraction (Davies et al.1992). BAC DNA was isolated using QIAGEN's midi prep kit. Total RNA wasisolated using either QIAGENS's Plant RNeasy Kit or the Trizol Reagent(Invitrogen, Carlsbad, Calif.).

Cloning of Plasmid Insert Flanking Sequences from the Tla1 Mutant

Genomic DNA of the tla1 mutant was digested with Apa I (New EnglandBiolab, Beverly, Mass.) and size-fractionated by 0.8% agarose gelelectrophoresis. Restriction enzyme digestion yielded a DNA fragment ofabout 5 kb, containing about 2 kb of Chlamydomonas genomic DNA flankingthe insertion and about 3 kb portion of the 5′ end of the pJD67 plasmidsequence (Polle et al. 2003, supra). Similarly, a 3.2 kb DNA fragmentwas identified, which contained about 2.2 kb of the 3′ end of the pJD67plasmid sequences and about 1 kb of Chlamydomonas genomic DNA flankingthe insertion (Polle et al. 2003, supra). Therefore, following agarosegel electrophoresis of Apa I-digested tla1 genomic DNA, fragmentsmigrating in the 6-4 kb region were used for the construction of a 5′insert flanking DNA library. Similarly, fragments migrating in the 4-3kb region were used to construct a 3′ insert flanking DNA library.

The vector was digested with Apa I and treated with alkaline phosphatase(Promega, San Luis Obispo, Calif.) to avoid self-ligation of theplasmid. DNA fragments that migrated between the molecular weightmarkers of 4-6 kb and 3-4 kb were gel purified using QIAEX II gelextraction kit (QIAGEN, Valencia, Calif.) and were ligated into thepZero Kan+ vector (which includes a kanamycin resistance gene, Promega,San Luis Obispo, Calif.) for the construction of a partial genomiclibrary containing insert-5′ and 3′ flanking sequences. Use of the Apa Irestriction enzyme in the digestion of the tla1 genomic DNA proveduseful not only in the spatial separation of the 5′-insert flankingsequences from the 3′-insert flanking sequences, but also in theseparation of the insert flanking sequences from the endogenous copy ofthe ARG7.8 gene.

About 5000 E. coli colonies containing the partial genomic DNA librariesof the tla1 mutant were screened using appropriate DNA probes derivedfrom the ARG7.8 gene (Polle et al. 2003, supra). The probe Sal I-Sal I(1.3 kb representing the 5′ end of the ARG7 structural gene) was used toscreen a partial genomic library containing the 5′-insert flankingsequences. The probe Nde I-Nde I (0.75 kb) derived from the 3′ end ofthe ARG7 was used to screen a partial genomic library containing the3′-insert flanking sequences (FIG. 1).

Isolation of a BAC Clone

A DNA fragment containing the insert-3′ flanking sequence wassubsequently used for screening a commercially available wild typeChlamydomonas reinhardtii BAC library, constructed in pBACmn vector andprinted on nylon membrane referred to as a high-density filter (IncyteGenomics Inc, Palo Alto, Calif.).

Southern Blotting and RACE Analysis

For Southern blot analysis, 10 μg of genomic or BAC DNA was used forrestriction digestion, separated on 0.8% agarose gels for Southern blotanalyses. After separation of the DNA fragments, nucleic acids wereeither blotted onto a positively charged nylon membrane (NEN LifeScience Products, Inc, Wellesley, Mass.) or DNA was purified fromexcised gel pieces in the region of appropriate molecular weight. Theblotted membranes were hybridized with ³²P-labeled probes (Randomoligonucleotides DNA Labeling System, Roche Diagnostic Corporation,Alameda, Calif.). The probe DNA was PCR-amplified using specific primersdesigned from the insert-3′ flanking sequence of the tla1 mutant. Bothof these primers were derived from the coding region of the Tla1 gene.

Tla1-cDNAs were synthesized using 1 μg of total RNA isolated from eitherWT or tla1 mutant with “primer 5” (Table 1) designed from the coding-2region of the Tla1 gene and an oligo dT anchor primer (Invitrogen,Carlsbad, Calif.). These cDNAs were used as templates for 5′ and 3′ RACEanalyses using a kit from Boehringer, Mannheim (Germany).

TABLE 1 Exemplary PCR primers and expected product sizes in wild type,tla1 mutant and tla1 complemented strains. Expected products fromstrains tla1 tla1- Primers WT mutant complements “primer1” (5′ TACGGGAATTTGCGGAACCTC 3′; SEQ 589 bp No product 589 bp ID NO: 4)and “primer 4” genomic genomic (5′TTGTTGTCCAGCACCAGCAC 3′; SEQ ID NO:5); DNA DNA product probing for the 5′UTR of Tla1 product “primer7” (5′ CAACGCATATAGCGCTAGCAG C 3′; No 681 bp 681 bp SEQ ID NO: 6) and“primer 4” (5′ product genomic genomic TTGTTGTCCAGCACCAGCAC 3′; SEQ IDNO: 5); DNA DNA product probing for the 3′end of pJD67 product “primer1” (5′ TACGGGAATTTGCGGAACCTC 3′; SEQ 939 bp No product 939 bp ID NO: 4)and “primer 6” (5′ AACACACACCCCGCACT genomic genomic 3′; SEQ ID NO: 31);probing for the full length Tla1 gene DNA DNA product; and transcriptproduct; 823 bp cDNA 823 bp product cDNA product “primer8” (5′GGGACTTCGTGGAGGACG 3′; SEQ ID No No product 436 bp NO: 8) andproduct genomic “primer 9” (5′ GGTTAGTCCTGCTCCTCGG 3′; SEQ ID DNAproduct NO: 9); probing for the Ble gene “primer3” (5′ GGGCCCTTCAGCTGCTCCGCTGACC 409 bp 40 bp 409 bp cDNA AAACC 3′; SEQID NO: 10)and “primer 5” (5′ cDNA cDNA product GGGCCCGAACGGGTTGTCCGCCTGCGCCTTGC 3′; product product SEQ ID NO: 11); Probing for theTla1 transcript “primer 2” (5′ GCTGCTCCGCTGACCAAA 3′; SEQ ID 525 bp 525bp 525 bp NO: 12) and “primer 5” (5′ genomic genomic genomicGGGCCCGAACGGGTTGTCCGCCTG CGCCTTGC 3′; DNA DNA DNA product; SEQ ID NO:11); Probing for the Tla1 transcript product; product; 454 bp cDNA 454bp no cDNA product cDNA product product TCF(5′ CGGGGTACCACTTTCAGCTGCTCCGCT 3′; SEQ ID NO: 13) and TCR (5′CCAAGCTTCCTCTT TCCCCCCCACC 3′; SEQ ID NO: 14); cloning primers used foramplifying the cDNA coding for the full length Tla1, off the cDNAlibrary for Tla1 overexpression. PCR product size is 750 bp.

Transformation of the Tla1 Mutant

BAC clone 39e16 DNA was digested with restriction enzymes ApaI or PstI.An approximately 3.7 kb DNA fragment, derived upon Apa I digestion (ApaI-Apa I), and an about 3 kb DNA fragment, derived upon Pst I digestion(Pst I-Pst I) were subcloned. The 2 kb DNA overlap region between thesetwo clones (p5′TlaApa-4 and p3′TlaPst-3-3) was removed upon Apa Idigestion of the 3 kb p3′TlaPst-3-3 clone. Subsequently, the Apa I -ApaI fragment and the 3′ end of the Pst I-Pst I DNA fragment, whichresulted from the ApaI digestion, were re-ligated to yield the complete4.7 kb sequence of the Tla1 gene in pBluescript. The resulting pFT1a-5plasmid DNA was sequenced to confirm the correct coding sequence of theTla1 gene.

The ble gene encoding zeocin resistance along with its RbcS2 promoterand terminator was excised from plasmid pSP124S (Stevens et al., Mol.Gen. Genet. 251: 23-30, 1996) by Hind III digestion and inserted at the5′ end of the Tla1 gene in tandem to generate plasmid pSK9.2B1eFT1a.This plasmid was linearized upon digestion with Kpn I and used totransform the tla1 mutant by the glass bead method (Debuchy et al., EMBOJ 8: 2803-2809, 1989). Transformant colonies were selected for zeocinresistance on TAP agar plates in the presence of 5 μM zeocin.Zeocin-resistant colonies were further screened for tla1 complementationby visual inspection of the colony coloration. Zeocin-resistant colonieshaving dark green coloration were tested for the presence of the wildtype Tla1 gene and Tla1 protein amount by PCR/RT-PCR and Western blotanalysis, respectively.

PCR and RT-PCR Analysis

Strains with tla1 mutations complemented with the Tla1 gene(tla1-complements) were first tested by PCR to check for the presence oftwo distinct Tla1 genes (wild type and mutant) and of the Ble tag. PCRwas applied to genomic DNA by using two different forward primers,namely a Tla1 5′UTR specific primer (“primer 1”: 5′TACGGGAATTTGCGGAACCTC 3′ (SEQ ID NO:4), Table 1) and a primer designedfrom the 3′ end of the pJD67 vector that was inserted just upstream ofthe start codon of the Tla1 gene (“primer 7”: 5′ CAACGCATATAGCGCTAGCAGC3′ (SEQ ID NO:6), Table 1). A reverse primer was defined from the secondexon of the Tla1 gene (“primer 4”: 5′ TTGTTGTCCAGCACCAGCAC 3′ (SEQ IDNO:5), Table 1). Upstream 5′UTR specific primer “primer 1” and the downstream 3′ UTR specific PCR primer (“primer 6”: 5′ AACACACACCCCGGCACT 3′(SEQ ID NO:31, Table 1) were used to probe for the full-length Tla1transcript. Tla1 5′UTR specific “primer 2” (5′ GCTGCTCCGCTGACCAAA 3′;SEQ ID NO:12) or Tla1 exon 1-specific “primer 3” (5′GGGCCCTTCAGCTGCTCCGCTGACCAAACC 3′ (SEQ ID NO:10, Table 1) were used inconjunction with the reverse “primer 5” (5′GGGCCCGAACGGGTTGTCCGCCTGCGCCTTGC 3; SEQ ID NO:11) defined from thesecond exon of the Tla1 gene to check for the presence of the Tla1transcript in the tla1-complemented strains. Presence of the Ble tag wastested by PCR using a forward primer located in the second exon of theBle gene (“primer 8”: GGGACTTCGTGGAGGACG 3′ (SEQ ID NO:8), Table 1) anda reverse primer located in the third exon of the Ble gene (“primer 9”:5′ GGTTAGTCCTGCTCCTCGG 3′ (SEQ ID NO:9), Table 1). The one-step RT-PCRkit (QIAGEN, Valencia, Calif.) was used for RT-PCR experiments. PlatinumTaq polymerase (Invitrogen, Carlsbad, Calif.) was used for the PCRamplification. A 1 kb plus DNA ladder was used as DNA size markers(Invitrogen, Carlsbad, Calif.).

Generation of Tla1 Protein Overexpression Constructs

An amplified Chlamydomonas cDNA core library obtained from thelaboratory of Dr. James V. Moroney (Louisiana State University, BatonRouge, La.) was used to amplify Tla1 cDNA for generating the Tla1protein overexpression construct. This cDNA library has been generatedby cloning the cDNA core library (Chlamydomonas Genetics Center, DukeUniversity) into the lambda ZapII vector (Stratagene, La Jolla, Calif.).In vivo excision of the pBluescript phagemid from the lambda ZapIIvector, involving the Ex-Assist interference-resistant helper phagealong with the SOLR strain of E. coli was used in the amplification ofthe cDNA core library.

The Tla1 cDNA sequence coding for the full length Tla1 protein wasamplified from the cDNA library by PCR. The 5′ end PCR primer (TCF) hasthe sequence 5′-CGGGGTACCACTTTCAGCTGCTCCGCT-3′ (SEQ ID NO:13) (Table 1)and a KpnI site was incorporated at the 5′ end. The 3′ end PCR primer(TCR) has the sequence 5′-CCCAAGCTTCCTCTTTCCCCCCCACC-3′ (SEQ ID NO:32)and a HindIII site was incorporated at the 5′ end. Amplified Tla1 cDNAswere purified from the DNA gel using the QIAEX II gel extraction kit(QIAGEN, Valencia, Calif.) and were cloned into the pQE80Loverexpression vector (QIAGEN, Valencia, Calif.) which has a 6*His (SEQID NO:33) tag. The vector and the purified Tla1 cDNAs were doubledigested with Kpn I and Hind III. Ligation of the 733 bp Tla1 PCRproduct immediately downstream of the 6*His (SEQ ID NO:33) tag sequencein the pQE80L vector was performed following the protocol given in theNew England Biolab (NEB, Beverly, Mass.) technical manual. E. colistrain DH5α cells were transformed. Transformants were isolated byscreening colonies on LB+Amp (100 μg/mL-1) plates. In-frame insertion ofTla1 with the His tag sequence in the recombinant clone was verified bydouble restriction enzyme digestion analyses with Kpn I and Hind III andDNA sequencing.

Overexpression and Purification of his-Tagged Tla1 Protein for theGeneration of Polyclonal Specific Antibodies

Selected E. coli clones of Tla1 were grown at 37° C. in 200 mL of LBmedia on a rotary shaker. The cells were induced for 5 h with 1 mM IPTGwhen the culture OD600 was between 0.6 and 0.7. Both induced anduninduced E. coli cells were harvested and resuspended in lysis buffer[100 mM NaH₂PO₄, 10 mM Tris-Cl (pH 8), 8 M urea] followed by sonication.Equal amounts of protein samples from induced and uninduced cells weresubjected to 12.5% SDS-PAGE gel electrophoresis to test for theoverexpression of the recombinant protein.

The recombinant fusion protein was purified by a one-step affinitychromatography using Ni-NTA superflow columns. Crude sonicated cellextracts were passed through Ni-NTA superflow columns (1 mL of thenickel-charged resin binds 10-15 mg of the recombinant protein). Thecolumn was washed with 6 L of wash buffer [100 mM NaH₂PO₄, 10 mMTris-HCl (pH 6.3), 8 M urea]. At the final step, fusion proteins wereeluted from the column by elution buffer [100 mM NaH₂PO₄, 10 mM Tris-HCl(pH 4.5), 8 M urea]. Purified recombinant proteins were furtherconcentrated by a passage through Centricon columns (Amicon, Billerica,Mass.). The recombinant proteins were recovered from the membrane of thefilter upon elution with phosphate buffered saline (pH 7.4) containing137 mM NaCl, 2.7 mM KCl, 4.3 mM NaH₂PO₄ and 1.4 mM KH₂PO₄4. Purificationof the recombinant protein was tested upon SDS-PAGE using the Benchmarkprestained protein ladder (Invitrogen, Carlsbad, Calif. The purifiedrecombinant protein was used for the generation of specific polyclonalantibodies (ProSci Incorporated (Poway, Calif.) following a standardprotocol. Approximately 1.6 mg of protein in each of two rabbits wasused to generate the Tla1 antibodies.

Cellular Protein Analysis

Chlamydomonas cells were harvested, washed twice with fresh medium andresuspended in TEN buffer (10 mM Tris-HCl, 10 mM EDTA and 150 mM NaCl;pH 8). Following sonication, the crude cell extract was incubated in thepresence of solubilization buffer (Smith et al. 1990). Proteinconcentration was determined and gel lanes were loaded with an equalamount of Chl, in the range of 4 to 6 nmol Chl, as indicated. SDS-PAGEanalysis was performed on a 12.5% gel, using either the Benchmarkprestained or unstained protein ladder (Invitrogen, Carlsbad, Calif.),at a constant current of 10 mA for 5 h. Gels were stained with 1%Coomassie brilliant Blue R for protein visualization.

Western Blot Analysis

Electrophoretic transfer of the SDS-PAGE resolved proteins ontonitrocellulose was carried out for 2 h at a constant current of 400 mAin the transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol).The titer of the Tla1 immune serum was probed with different amounts ofthe purified recombinant His tagged Tla1 protein (2 pg-20 ng), as wellas with the total protein extract of wild type (CC425), tla1 mutant, andtla1-complements. The Tla1 immune serum was diluted with buffer[Tris-buffered saline, 0.005% Tween 20 and 1% bovine serum albumin (pH7.4)] to a ratio of 1:3,000 before being used as a primary probe. Thesecondary antibody used for Western blotting was conjugated tohorseradish peroxidase (BioRad, Hercules, Calif.) and diluted to a ratioof 1:30,000 with the antibody buffer. Western blots were developed byusing The Supersignal West chemiluminescent substrate kit (Pierce,Rockford, Ill.).

Accession Numbers

GenBank Accession numbers for the exemplary Tla1 sequences in theexamples are AF534570 (complete Tla1 genomic DNA sequence with exons andintron) and AF534571 (complete mRNA sequence with 5′ and 3′ untranslatedregions).

Example 1 Cloning of the Tla1 Gene

Southern blot analyses of the Chlamydomonas reinhardtii tla1 mutantrevealed a single pJD67 plasmid insert in the nuclear genome. Geneticcrosses and random progeny analyses revealed that the exogenous ARG7.8gene co-segregated with the tla1 phenotype (Polle et al. 2003, supra).On the basis of these properties, it was inferred that insertion of thepJD67 plasmid must have interrupted, or deleted, a gene that is involvedin the regulation of the light-harvesting Chl antenna size ofphotosynthesis.

The 5′ end vector sequence information, required for plasmid rescue, hadbeen deleted from the insert site (FIG. 1A). Thus, plasmid rescue couldnot be employed for the cloning of the genomic DNA that is flanking theinsert. To identify the gene, two different partial genomic libraries ofthe tla1 mutant, representing the 5′ and 3′ plasmid insert flankingsequences were constructed and screened with appropriate probes (seeMaterials and methods). Three positive clones were identified from the5′ insert flanking DNA library and only one positive clone from the 3′insert flanking DNA library. All of the above four positive clones wereconfirmed by restriction enzyme and sequence analysis.

A database search with the 5′-insert flanking sequence did not showsignificant homology to any existing EST sequences. However, a BLASTsearch with the 3′-insert flanking sequence matched the ChlamydomonasEST sequence 894001DO4.yl, which was deposited in the GenBank withAccession No. BE024188. The designation ‘y’ in this EST sequence denoteda 5′ end of the respective cDNA.

Isolation of a BAC Clone Containing the Full Length Tla1 Gene

The 3′-insert flanking sequence of tla1 was used to screen ahigh-density filter containing a Chlamydomonas wild type BAC library. ABAC clone, number 39e16, was identified as containing the complete Tla1gene sequence. Southern blot analysis of the 39e16 clone DNA withseveral restriction enzymes (using the 3′-insert flanking sequence as aprobe) permitted construction of a Tla1 restriction map.

FIG. 1A shows a map of the pJD67 insertion site in the nuclear genome ofthe tla1 mutant. It is shown that about 2.3 kb of the 5′ end of thepJD67 was deleted upon plasmid insertion in the C. reinhardtii nucleargenome. It is also shown that about 6 kb of genomic DNA was deleted uponplasmid insertion. Nine bps of the 3′ end of the plasmid sequences werealso deleted upon plasmid insertion.

A full-length cDNA was obtained upon RT-PCR, and 5′ and 3′ RACE analysesusing cDNA from the WT (cw15) strain. The full-length cDNA showed anopen reading frame encoding a protein of 213 amino acids. DNA sequenceanalyses of the 39e16 BAC clone revealed the structure of thefull-length Tla1 genomic DNA. Genomic and cDNA analysis of the Tla1 geneshowed the presence of a 104 bp 5′ UTR, a single intron of 116 bases,and 1.26 kb of 3′ UTR. FIG. 1B also compares the DNA structure in theTla1 upstream region in wild type and tla1 mutant. In the tla1 mutant,the 3′ end of the pJD67 plasmid replaced the promoter and 5′ UTR of thewild type gene.

Transcription of the Tla1 gene in wild type and tla1 mutant was testedby RT-PCR and compared to that of genomic DNA PCR. Given the presence ofthe pJD67 insert in the 5′ UTR of the Tla1 gene, a question was raisedas to whether the tla1 mutant was able to transcribe the remnant of theTla1 gene. RT-PCR was performed with a forward primer designed from the5′ UTR sequence of Tla1, (“primer 2”) and a reverse primer (“primer 5”)from the coding-2 sequence of this gene (FIG. 2). This RT-PCR yieldedproducts in the WT but not in the tla1 mutant (FIG. 3, lanes 1 and 2).With the set of PCR primers that were designed from within the codingsequence of the Tla1 (“primer 3” and “primer 5”), RT-PCR yieldedproducts in both the WT and tla1 mutant (FIG. 3, lanes 4 and 5). PCRproducts obtained with the same primers from the genomic DNA of the tla1strain were 116 bp larger than those obtained from the cDNA due to thepresence of an intron (FIG. 3, lane 6).

5′ Race analysis of the Tla1 cDNAs from the wild type and mutantrevealed polymorphism in the 5′ UTR sequences of wild type and tla1mutant. FIG. 4 (upper) shows the 5′ RACE analysis of the wild type cDNAwith a portion of the 5′ UTR, the ATG start codon, and the correspondingdownstream coding nucleotide sequence. In the tla1 mutant (FIG. 4,middle panel), the ATG start codon is preserved. However, the entireupstream 5′ UTR and promoter regions of the Tla1 gene are deleted andreplaced by the 3′ end of the pJD67 plasmid sequence, represented by thelower case and underlined nucleotide sequence. FIG. 4 (lower panel)shows the nucleotide sequence of the complete 3′ end of the pJD67plasmid DNA. Nucleotides denoted in upper case characters belong to theARG7.8 gene. Nucleotides denoted in lower case characters belong to the3′ end of the vector sequence (pBR322). The underlined portion of thepJD67 3′end is also found in the cDNA nucleotide sequence obtained fromthe 5′ RACE analysis of the tla1 mutant (FIG. 4, middle panel, lowercase underlined nucleotides). Note that nine bases, i.e., at t a a a g ct, at the 3′ end of the full-length pJD67 (FIG. 4, lower panel) weredeleted from the insertion site (FIG. 4, lower panel, black background).In sum, 187 bp of the 3′ end of the pJD67 vector sequence have becomethe 5′ UTR sequence of the Tla1 gene in the tla1 mutant, as they wereamplified by the 5′ RACE in the latter.

Complementation of the Tla1 Mutant

A 4.7 kb genomic DNA, representing the full length Tla1 gene was clonedin pBluescript (FIG. 5). This plasmid was digested with Hind III and theBle gene was added at the 5′ end of the Tla1 gene to generate plasmidpSK9.2B1eFTla1 (see Materials and methods). This plasmid was used tocomplement the tla1 mutant. Transformant colonies were selected on agarplates in the presence of 5 μM zeocin and screened for dark greencoloration. Three dark green colonies, putative complements of the Tla1gene were randomly isolated and streaked on to a TAP agar plate alongwith the wild type and tla1 mutant strains (FIG. 6). These putativecomplements, tla1-comp1, tla1-comp2 and tla1-comp3, showed wild typephenotype in terms of coloration and in vivo chlorophyll fluorescenceinduction kinetics. The putative complements were grown autotrophicallyin TBP medium and tested for the Chl/cell and Chl a/Chl b ratios.

Table 2 shows that wild type C. reinhardtii had a Chl a/Chl b ratio of2.6, whereas the tla1 mutant had a Chl a/Chl b ratio of 6. The putativetla1-complemented strains had much lower Chl a/Chl b ratios, rangingbetween 2.8-3.0. These values are much closer to that of the wild typethan to the tla1 parental host strain. A lower Chl a/Chl b ratiosuggests assembly of peripheral subunits of the Chl a-b light-harvestingcomplex, underlying an enlarged photosystem Chl antenna size (Polle, etal., Plant Cell Physiol. 42: 482-491, 2001; and Polle et al., 2003,supra). Table 2 also shows the Chl/cell values of the various strains.Chl/cell in the wild type (3.2×10-15 mol/cell) was substantially greaterthan that in the tla1 mutant (1.1×10⁻¹⁵ mol/cell). The complementedstrains had Chl/cell values (2.2-2.8×10⁻¹⁵ mol/cell) comparable to thatof the wild type, providing evidence of the return of thetla1-complemented strains to wild type levels of Chl content. Moreover,chlorophyll fluorescence induction kinetic measurements, with intactcells in the presence of DCMU, showed that the complemented strains,very much like the wild type, had a sigmoidal fluorescence rise curve,evidence of a statistical pigment bed organization afforded by a largeChl antenna size, as compared with the exponential fluorescenceinduction kinetics in the tla1 mutant (not shown). The lower Chl a/Chl bratio, greater Chl/cell ratio (Table 2) and sigmoidal fluorescenceinduction kinetics in the Tla1-complemented strains are evidence of adirect cause-and-effect relationship between the amount of the Tla1protein and the amount of chlorophyll in C. reinhardtii. Thus, it isconcluded that the Tla1 gene regulates the Chl antenna size ofphotosynthesis in this model green alga.

TABLE 2 Chl a/Chl b ratio and Chl content per cell in C. reinhardtiiwild type (WT), tla1 mutant and tla1 mutant complemented with the Tla1gene (comp1-3). Statistical error (+/−SD) was <10% of the values shown.Chl a/Chl b Chl, ×10⁻¹⁵ Strain ratio mol/cell WT (cw15) 2.6 3.2 tla1 6.01.1 tla1-comp1 2.9 2.8 tla1-comp2 3.0 2.2 tla1-comp3 2.8 2.4

Example 2 Tla1 Gene Expression is Reduced in the Tla1 Mutant Strain

In the subsequent more detailed biochemical and molecular analyses, acomparative and quantitative evaluation of wild type, tla1 mutant,tla1-comp1 and tla1-comp2 was undertaken. When PCR was performed on thegenomic DNA using “primer 1” and “primer 4” (Tla1 5′UTR/Exon-2, FIGS. 2Aand 2C) wild type, tla1-comp1 and tla1-comp2 yielded a 589 bp product,whereas the tla1 mutant failed to yield a product, consistent with theabsence of its 5′UTR region (FIG. 7A). When “primer 7” and “primer 4”(pJD67 3′ end/Tla1 Exon-2, FIG. 2B) were used in the PCR reaction, thetla1 mutant, tla1-comp1 and tla1-comp2 generated a product of 684 bpwhereas the wild type did not, consistent with the absence of the pJD67plasmid in the latter (FIG. 7B). When Ble primers, “primer 8” and“primer 9” (FIG. 2C) were used for the genomic DNA PCR reaction,tla1-comp1 and tla1-comp2 yielded a product of 436 bp, whereas both wildtype and tla1 mutant failed to generate a product (FIG. 7C). PCR wasalso employed to test for the presence of the intact full length Tla1gene in the two complements using the “primer 1” and “primer 6” (Tla15′UTR/Tla1 3′UTR, FIG. 2A). Wild type, tla1-comp1 and tla1-comp2 gave aproduct of 939 bp whereas the tla1 mutant did not yield a product (FIG.7D). When the same primers were used to perform RT-PCR, wild type,tla1-comp1 and tla1-comp2 gave a product of 823 bp, whereas the tla1mutant did not yield a product (FIG. 7E). These results are evidence ofsuccessful complementation of the tla1 mutant by the ble-Tla1 construct.

Western Blot Analysis with Tla1-Specific Antibodies in Wild Type, Tla1Mutant and Tla1 Complemented Strains

E. coli cells harboring the recombinant 6*His-Tla1 construct wereinduced for 5 h at 37° C. to overexpress the His-tagged Tla1 fusionprotein. The overexpressed recombinant Tla1 protein comprisedapproximately 20% of the total E. coli protein (FIG. 8A). Therecombinant fusion protein was purified by one-step affinitychromatography using Ni-NTA superflow columns and further concentratedby a passage through Centricon columns. Purification of the 25 kD fusionprotein was confirmed by SDS-PAGE and Coomassie staining (FIG. 8B).

Tla1 specific polyclonal antibodies positively cross-reacted with thepurified 25 kD recombinant Tla1 protein and at levels as low as 2 pg(FIG. 9A). Whereas the molecular weight of the native Tla1 protein is23.2 kD, the recombinant protein was slightly larger (25 kD) because ofthe extra seventeen amino acids, including the 6*His tag (SEQ ID NO:33),at its N-terminal end.

Total cell extract from wild type (CC425) and the tla1 mutant wereloaded on a 12.5% SDS-PAGE gel on an equal chlorophyll basis (FIG. 9B).Tla1 antibodies detected the 23.2 kD Tla1 protein in the total proteinextract from the wild type and tla1 mutant (FIG. 9C). The amount of theTla1 protein was substantially lower in the tla1 mutant compared to thatin the wild type (FIG. 9C). This provides evidence that a limitedtranslation of the tla1 mRNA did occur in the mutant, however, this isin no way comparable to the levels of translation seen in the wild type.The substantially suppressed translation level of the Tla1 protein inthe tla1 mutant is attributed to the absence of the native 5′UTR in thisstrain. The apparent molecular weight of the Tla1 protein is the same inWT and tla1 mutant (FIG. 9C). This observation is consistent with thenotion that the wild type Tla1 protein lacks a transit peptide and isapparently a cytoplasmic protein.

Total protein extracts from the wild type, tla1 mutant and two tla1complements (tla1-comp1 and tla1-comp2) were resolved on a 12.5%SDS-PAGE gel, lanes loaded on an equal chlorophyll basis (FIG. 10A).Western blot analysis of the total cell extract from these samplesshowed that the amount of the Tla1 protein in the two complements (FIG.10B, lanes 1 and 2) was comparable to that in the wild type (FIG. 10B,W), whereas the amount of the Tla1 protein in the tla1 mutant wassubstantially lower from that of the other three (FIG. 10B, lane T).

Example 3 Analysis of Tla1 Protein Hydropathy Analysis of the Tla1Protein

Analysis of the N-terminus sequence of the predicted Tla1 protein byChloroP, (http://www.cbs.dtu.dk/services/ChloroP/), TargetP(http://www.cbs.dtu.dk/services/TargetP/) and MitoP(http://ihg.gsf.de/ihg/mitoprot.html) software programs failed toindicate the presence of a transit peptide, suggesting that Tla1 is acytosolic protein. This indication was strengthened by the results ofthe Western blot analysis, where the mature protein size matched thepredicted translation product size, suggesting absence of a cleavabletransit peptide. FIG. 11 shows the hydropathy plot of the deduced aminoacid sequence of the Tla1 protein, derived according to the method ofKyte & Doolittle (Kyte and Doolittle, J Mol Biol. 157:105-32982, 1982;http://occawlonline.pearsoned.com/bookbind/pubbooks/bc_mcampbell_genomics_(—)1/medialib/activities/kd/kyte-doolittle.htm).The Tla1 protein contains 213 mostly hydrophilic amino acids, suggestingthat it is a soluble cytosolic protein. There was a single hydrophobicdomain comprising 27 amino acids between residues 42 and 69,theoretically long enough to qualify as a transmembrane domain. Thishydrophobic domain of 27 amino acids is highly conserved in similarproteins from other diverse organisms, suggesting a role in thecatalytic/regulatory activity of the Tla1 protein.

Tla1 Homology with Genes from Other Organisms

A Blastp (protein database using protein sequence) search showed highhomology of the Tla1 protein with expressed protein sequences ofArabidopsis thaliana (GenBank Accession No. NP_(—)568832) Oryza sativa(japonica cultivar-group) (Accession No. CAD39888) Ustilago maydis(Accession No. EAK83164), Drosophila melanogaster (Accession No.NP_(—)611731), Homo sapiens (Accession No. AAQ83690), Danio rerio(zebrafish) (Accession No. NP_(—)956420), Rattus norvegicus (Norway rat)(Accession No. XP_(—)214198), Xenopus tropicalis (Accession No.NP_(—)989181), Mus musculus (house mouse) (Accession No. NP_(—)035056).In view of the Chlamydomonas reinhardtii specific codon usage, we alsosearched nucleotide databases using the Tla1 protein sequence(tblastn—protein query against translated data base). EST sequencesdeposited from several other plant species showed fairly high homologywith the Tla1, including sequences from Hordeum vulgare, Solanumtuberosum, Medicago truncatula, Lycopersicon esculentum, Gossypiumarboretum, Secale cereale, Triticum aestivum, Pinus taeda, Betavulgaris, Populus tremula, Sorghum bicolor (results not shown). It isconcluded that the Tla1 gene is present in many eukaryotes, includingmany wild-land and crop plants.

FIG. 12A shows an alignment of the deduced amino acid sequence of theTla1 protein from C. reinhardtii alongside that of proteins from A.thaliana, O. sativa, H. sapiens and D. melanogaster. The sequencealignment in FIG. 12A shows very high similarity between the Tla1protein in C. reinhardtii and the related proteins in these diverseorganisms. Moreover, examination of identity/similarity patterns in FIG.12A, based on the ClustalW analysis, revealed the common occurrence ofdomains where high similarity or identity of amino acids is observed.The highly conserved nature of these domains across diverse speciessuggest a common functional role for these proteins.

These related protein sequences were also aligned pair-wise and degreesof identity, high and low similarity were calculated on the basis of aClustalW comparison. Results from such analyses (Table 3) showed thatthe C. reinhardtii Tla1 protein had a 72.68% homology to thecorresponding protein in A. thaliana, 75.99% homology to O. sativa,70.94% to D. melanogaster and 67.14% to H. sapiens (CGI-112). FIG. 12Bshows a phylogenetic tree of the above-mentioned Tla1 homologues, basedon the amino acid sequence comparisons (www.ebi.ac.uk/clustalw/).

TABLE 3 Homology comparison of Tla1-like proteins in Chlamydomonasreinhardtii, Arabidopsis thaliana, Oryza sativa, Drosophila melanogasterand Homo sapiens. The % of amino acid identity, high similarity and lowsimilarity was based on a ClustalW analysis of the deduced amino acidsequence of the respective proteins. High Low Pair compared % Identitysimilarity similarity % Homology C.r.-A.t. 35.62 23.75 13.31 72.68C.r.-O.s.. 38.27 23.92 13.8 75.99 C.r.-D.m. 30.54 23.15 17.24 70.94C.r.-H.s. 28.09 24.76 14.28 67.14

SUMMARY

The ability of the photosynthetic apparatus to regulate the size of thefunctional Chl antenna was first recognized in pioneering work byBjorkman and co-workers, more than 30-years ago (Bjorkman et al.,Carnegie Institution Yearbook 71:115-135, 1972). In spite of thesubstantial number of physiological and biochemical studies on thisphenomenon (reviewed Anderson, Annu Rev Plant Physiol 37: 93-136, 1986;Melis, In, Oxygenic Photosynthesis: The Light Reactions” (D R Ort, C FYocum, eds), Kluwer Academic Publishers, Dordrecht, The Netherlands, pp.523-538, 1996), genes for the regulation of the Chl antenna size ofphotosynthesis have not been identified. The current invention is basedon the discovery that Tla1 plays a role in the regulation of thechlorophyll antenna size of photosynthesis. Not to be bound by theory,Tla1 is likely to regulate the expression of other genes that directlyaffect the chloroplast and the Chl antenna size and may define therelationship between nucleus and organelle in green algae, therebyregulating the rate of Chl biosynthesis and by extension the Chl antennasize of the photosystems. For example, in the tla1 mutant, total amountof Chl, Lhcb gene expression, abundance of LHC polypeptides, and levelsof Chl b were all down regulated (Table 2), presumably as a consequenceof down-regulation of translation of the Tla1 mRNA. Sensitiveabsorbance-difference kinetic spectrophotometry confirmed that the tla1mutant had a truncated PSII Chl antenna size, down to 50%, and atruncated PSI Chl antenna size, down to 67% of that in the wild type(Polle et al. 2003, supra). Thus, in the tla1 mutant, the Chl antennasize of both photosystems, as well as total chlorophyll per cell werelowered relative to the wild type.

Further evidence for the role of the Tla1 gene in the regulation of theChl antenna size of photosynthesis was obtained from the study of C.reinhardtii diploid analysis and from Tla1 complementation studies.Diploid analysis showed that the tla1 mutation is recessive (results notshown). About 25 diploids were tested, all of which showed WT phenotypein terms of normal green coloration of the colonies, normal Chlfluorescence and normal Chl a to Chl b ratio (in the range of 2.2-3.0)as opposed to the tla1 mutant phenotype of yellow-green coloration,lower Chl fluorescence and higher Chl a to Chl b ratio. The results ofthe diploid analysis served as the basis upon which a functionalcomplementation of the tla1 mutant was undertaken. This was successfullyimplemented (FIGS. 6 and 7) upon transformation of the tla1 strain witha WT copy of the Tla1 gene, containing about a 4.7 kb DNA sequencecomprising the promoter region and its 5′ flanking sequence, the 5′ UTR,the coding sequence with a single intron, and the 3′ UTR region of theTla1 gene.

Sequence analysis of the 3′-insert flanking sequence from the tla1mutant revealed that the 3′ end of the plasmid was inserted within theC. reinhardtii genomic DNA, just prior to the ATG start codon of theTla1 gene. In spite of the absence of the promoter and 5′UTR region ofthe Tla1 gene and the presence of a sizable plasmid insertion just priorto the ‘ATG’ start codon of the Tla1 gene, RT-PCR analysis (FIG. 3)revealed the presence of Tla1 transcripts in the tla1 mutant. Onepossible explanation of this unusual observation is that, in the tla1mutant, the Tla1 gene is co-transcribed along with the ARG7 gene underthe control of the ARG7 gene promoter. Northern blot analyses, using thecoding region of the Tla1 gene as a probe, revealed similar theTla1-transcript size from both WT and tla1 mutant (results not shown).High molecular weight transcripts of the Tla1 gene could not be detectedin the tla1 mutant, as would be expected from the unprocessed ARG7-Tla1hybrid transcripts.

From the above characteristics (tla1 phenotype, diploid properties,complementation of the tla1 mutant with wild type Tla1 gene, andpresence of Tla1 transcripts in the tla1 mutant), it is concluded thattranscription of the Tla1 gene occurs in the tla1 mutant but translationof the respective mRNA is either impaired or minimized Polymorphism inthe 5′ UTR of the Tla1 gene has not measurably affected its mRNAstability since levels of these transcripts detected by Northern blot(data not shown) and RT-PCR analysis were the same in both the wild typeand mutant. However, it has had a substantial effect on the rate oftranslation of the respective mRNAs, as evidenced from the Western blotanalysis results. In the tla1 mutant, the 5′UTR consists of 187 bpsequences from the 3′end of the plasmid pJD67. Normally, the eukaryotictranslation machinery does not recognize prokaryotic sequences. Thiscould explain the much lower translation levels of the Tla1 mRNA in themutant relative to that in the wild type.

It is apparent from these data that in green unicellular algae the Tla1gene acts as an early component affecting the molecular regulatorymechanism for the Chl antenna size in of oxygenic photosynthesis. Agenetic tendency of the algae to assemble large arrays of lightabsorbing chlorophyll antenna molecules in their photosystems is asurvival strategy and a competitive advantage in the wild, where lightis often limiting (Kirk, Light and photosynthesis in aquatic ecosystems,2nd edn. Cambridge University Press, Cambridge, England, 1994). Thisproperty of the algae is detrimental to the yield and productivity in amass culture under direct sunlight (Melis, Trends in Plant Science 4:130-135, 1999), however. A truncated Chl antenna size, which wouldcompromise the ability of the strain to compete and survive in the wild,is helpful in a controlled mass culture environment in photoreactors indiminishing the over-absorption and wasteful dissipation of excitationenergy by individual cells. The size reduction of the Chl antenna willalso diminish photoinhibition of photosynthesis (Powles, Annu Rev PlantPhysiol 35: 15-44, 1984; Melis, 1999, supra) at the surface whilepermitting for greater transmittance of light deeper into the culture(Melis, 2005, supra). Such altered optical properties of the cellsresult in greater photosynthetic productivity and enhanced solarconversion efficiency by the culture as a whole. In support of thiscontention, preliminary experiments (Powles, Annu Rev Plant Physiol 35:15-44, 1984) confirmed that a smaller Chl antenna size would result in arelatively higher light intensity for the saturation of photosynthesisin individual cells, while permitting for an overall greater solarconversion efficiency and productivity by the mass culture (Powles, AnnuRev Plant Physiol 35: 15-44, 1984). Thus, the Tla1 gene can be useful asa target to down-regulation Chl antenna size, e.g., in green microalgae.

The above examples are provided by way of illustration only and not byway of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

All publications, accession numbers, and patent applications cited inthis specification are herein incorporated by reference as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference.

Table of Exemplary Tla1 nucleic acid and polypeptide sequences SEQ IDNO: 1 Tla1 cDNA sequence--coding sequence 105-746 1 ggaacctcgatgtcgtgttg actttgcgtt acaaccgtga agtatattag aactcatttg 61 cctgccacaacctcagacca agagacgcgc gaaaaactga cacgatgact ttcagctgct 121 ccgctgaccaaaccgcgctc ttaaagattc ttgcacacgc ggctaagtat ccatcaaata 181 gcgtgaatggtgtcctcgtc gggacagcga aggagggcgg ctctgtcgaa atcctggacg 241 cgattccactgtgtcacacg acgctgaccc tggcgccagc actggagata ggtctcgccc 301 aggtggagtcctacacgcat atcacgggca gcgtggcgat tgtgggctac taccaatcag 361 acgcacgtttcggccccggg gacctacccc cgctaggtcg caaaattgcg gacaaggtgt 421 ctgagcaccaggctcaggcg gtggtgctgg tgctggacaa caagcggctg gagcagttct 481 gcaaggcgcaggcggacaac ccgttcgagc tgttcagcaa ggatggcagc aagggttgga 541 agcgcgcgagcgccgatggc ggagagctgg cgcttaaaaa cgcggactgg aagaagctgc 601 gcgaggagttcttcgttatg ttcaagcagc tgaagcaccg gacactccac gattttgagg 661 agcacctggacgacgccggg aaagactggc tcaacaaggg cttcgcctcc tcggtcaaat 721 tcctgttgcccggcaacgcg ctgtaagggc cgcgtgaggc tagccgggat ggcggttccg 781 cgggatggtcgcagtgccgg ggtgtgtgtt gagaggagga gccggtgggg gggaaagagg 841 ttgaggaggtaggagagagg cgctggcatg gaggccggga ggcgctggag ctggagctgg 901 cgagctggtgggtggtgctg ggcgagatcc tggaggcaca ggagtggtat gggcggtgca 961 gggacagcgacagcggatcg gcggacggta ttggtggagg gtgcgggggc cctggggtag 1021 tgtgcagggtgtgtgccacg tggcttgccg caaagcgcag cgtaccgata gttgagagaa 1081 agcacctgcggccctgcgcg gccgcggcgt ggcggcgcgt ggggacacgc gcatcgtgcc 1141 gggtcgccgcaggccggagt gaatttcgtg ctgcacggcg cgttgaccag tccaccgact 1201 gacggccaacggccatgagg gcttgttttg ggggataggg tcacatgaca ttttcggcgt 1261 tctttgcagtcagaatcagg atacgcttgc tttagtcttg attgtcagac ttgtcaggct 1321 gacgtttcaggcagacgaga gctcatgtgg ttttgactaa ccgggcgttg accatgggca 1381 gtcccaaacgtgccgtgcca cagggcatag cgagtgccat gtgctctcga gggcgaggtc 1441 gtgaggcacgtggaaactgt tgcggcgcct tcaccatggg tgctttctcg cgtgaggcac 1501 gtgaaactgttgcggcgcct tcaccatggg tgctctctct cgtgaggctc agcggcaagt 1561 accagggagggcgcaagaca cggatgaagc agtggttgcg catgccgcgg tctgttggcc 1621 gccgggaggtgatcggtgtg acgtggctgg tgcgtgtggt ggtttctccc gtggcctccc 1681 gtgtgtgactggtgcgtgtt tgacgtggca aggtaggtaa atagtagtaa agcggcccag 1741 atacgttgctgtggcggttg tgcgtgcgca ggtggtgcat aggacagcgt tggttgtgtg 1801 tgcctgtgctgtgctgtgcg gtgccggacc gaagcgcggg gcggacaggc gcagggtggt 1861 agcggcgtggcgggtaggct gccgcacaca gtacgtgtaa ctgtatgctg cgctgcatgt 1921 tactctgcttacggatgctt cctgactgta cgtgtggtgc ttgggtcgtg tcgccgtgca 1981 acgctgctggcggcttcaat gggtggctgc ggatcagtgg gtggctgcgt gtatcggcgc 2041 gcccgtgttgaatcgaggac tgcag SEQ ID NO: 2 Tla1 polypeptide sequenceMTFSCSADQTALLKILAHAAKYPSNSVNGVLVGTAKEGGSVEILDAIPLCHTTLTLAPALEIGLAQVESYTHITGSVAIVGYYQSDARFGPGDLPPLGRKIADKVSEHQAQAVVLVLDNKRLEQFCKAQADNPFELFSKDGSKGWKRASADGGELALKNADWKKLREEFFVMFKQLKHRTLHDFEEHLDDAGKDWLNKGFASSVKFLLPGNAL Conserved Domains (SEQ IDNOs: 24-28): Domain A: amino acid positions 9-33(QTALLKILAHAAKYPSNSVNGVLVG) Domain B: amino acid positions 41-70(VEILDAIPLCHTTLTLAPALEIGLAQVESY) Domain C: amino acid positions 75-129(GSVAIVGYYQSDARFGPGDLPPLGRKIADKVSEHQAQAVVLVLDNKRLEQFCKAQ) Domain D:amino acid positions 135-163 (ELFSKDGSKGWKRASADGGELALKNADWK) Domain E:amino acid positions 177-200 (KHRTLHDFEEHLDDAGKDWLNKGF) SEQ ID NO: 3Tla1 genomic sequence 1 ggaacctcga tgtcgtgttg actttgcgtt acaaccgtgaagtatattag aactcatttg 61 cctgccacaa cctcagacca agagacgcgc gaaaaactgacacgatgact ttcagctgct 121 ccgctgacca aaccgcgctc ttaaagattc ttgcacacgcggctaagtat ccatcaaata 181 gcgtgaatgg tgtcctcgtc gggacagcga aggagggcggctctgtcgaa atcctggacg 241 cgattccact gtgtcacacg acgctgaccc tggcgccagcactggagata ggtctcgccc 301 aggtgcgcat ggccccgaga gcccggggcg tggcttgtgctcgtcgatct gcgtgcatta 361 gttaccgcat cgctcccatg ctgcattccg cgctcagcctcaaataccct gattgcaggt 421 ggagtcctac acgcatatca cgggcagcgt ggcgattgtgggctactacc aatcagacgc 481 acgtttcggc cccggggacc tacccccgct aggtcgcaaaattgcggaca aggtgtctga 541 gcaccaggct caggcggtgg tgctggtgct ggacaacaagcggctggagc agttctgcaa 601 ggcgcaggcg gacaacccgt tcgagctgtt cagcaaggatggcagcaagg gttggaagcg 661 cgcgagcgcc gatggcggag agctggcgct taaaaacgcggactggaaga agctgcgcga 721 ggagttcttc gttatgttca agcagctgaa gcaccggacactccacgatt ttgaggagca 781 cctggacgac gccgggaaag actggctcaa caagggcttcgcctcctcgg tcaaattcct 841 gttgcccggc aacgcgctgt aagggccgcg tgaggctagccgggatggcg gttccgcggg 901 atggtcgcag tgccggggtg tgtgttgaga ggaggagccggtggggggga aagaggttga 961 ggaggtagga gagaggcgct ggcatggagg ccgggaggcgctggagctgg agctggcgag 1021 ctggtgggtg gtgctgggcg agatcctgga ggcacaggagtggtatgggc ggtgcaggga 1081 cagcgacagc ggatcggcgg acggtattgg tggagggtgcgggggccctg gggtagtgtg 1141 cagggtgtgt gccacgtggc ttgccgcaaa gcgcagcgtaccgatagttg agagaaagca 1201 cctgcggccc tgcgcggccg cggcgtggcg gcgcgtggggacacgcgcat cgtgccgggt 1261 cgccgcaggc cggagtgaat ttcgtgctgc acggcgcgttgaccagtcca ccgactgacg 1321 gccaacggcc atgagggctt gttttggggg atagggtcacatgacatttt cggcgttctt 1381 tgcagtcaga atcaggatac gcttgcttta gtcttgattgtcagacttgt caggctgacg 1441 tttcaggcag acgagagctc atgtggtttt gactaaccgggcgttgacca tgggcagtcc 1501 caaacgtgcc gtgccacagg gcatagcgag tgccatgtgctctcgagggc gaggtcgtga 1561 ggcacgtgga aactgttgcg gcgccttcac catgggtgctttctcgcgtg aggcacgtga 1621 aactgttgcg gcgccttcac catgggtgct ctctctcgtgaggctcagcg gcaagtacca 1681 gggagggcgc aagacacgga tgaagcagtg gttgcgcatgccgcggtctg ttggccgccg 1741 ggaggtgatc ggtgtgacgt ggctggtgcg tgtggtggtttctcccgtgg cctcccgtgt 1801 gtgactggtg cgtgtttgac gtggcaaggt aggtaaatagtagtaaagcg gcccagatac 1861 gttgctgtgg cggttgtgcg tgcgcaggtg gtgcataggacagcgttggt tgtgtgtgcc 1921 tgtgctgtgc tgtgcggtgc cggaccgaag cgcggggcggacaggcgcag ggtggtagcg 1981 gcgtggcggg taggctgccg cacacagtac gtgtaactgtatgctgcgct gcatgttact 2041 ctgcttacgg atgcttcctg actgtacgtg tggtgcttgggtcgtgtcgc cgtgcaacgc 2101 tgctggcggc ttcaatgggt ggctgcggat cagtgggtggctgcgtgtat cggcgcgccc 2161 gtgttgaatc gaggactgca g

What is claimed is:
 1. A method of decreasing chlorophyll antenna sizein a plant, the method comprising: inhibiting expression of a Tla1nucleic acid in a plant by introducing into the plant an expressioncassette comprising a promoter operably linked to a polynucleotide, or acomplement thereof, that specifically hybridizes to a nucleic acid thatis at least 70% identical to at least 200 contiguous nucleotides of theTla1 nucleic acid; and selecting a plant with decreased chlorophyllantenna size compared to a plant in which the expression cassette hasnot been introduced.
 2. The method of claim 1, wherein the promoter isconstitutive.
 3. The method of claim 1, wherein the promoter isinducible.
 4. The method of claim 1, wherein the polynucleotide isoperably linked to the promoter in the antisense orientation.
 5. Themethod of claim 1, wherein the polynucleotide is operably linked to thepromoter in the sense orientation.
 6. The method of claim 1, wherein theplant is green algae.
 7. A plant comprising an expression cassettecomprising a polynucleotide, or a complement thereof, that specificallyhybridizes to a nucleic acid that is at least 70% highest percentidentity to at least 200 contiguous nucleotides of a Tla1 nucleic acid.8. The plant of claim 7, wherein the plant is green algae.
 9. A methodof enhancing yields of photosynthetic productivity under high-densitygrowth conditions, the method comprising cultivating a plant of claim 7under bright sunlight and high density growth conditions.
 10. A methodof enhancing production of a compound of interest, the method comprisingsuppressing Tla1 gene expression in a plant; and cultivating the plantunder conditions in which the compound is produced.
 11. The method ofclaim 10, wherein the plant is green algae.
 12. The method of claim 11,wherein the compound is H₂, biodiesel, Beta-carotene, lutein,zeaxanthin, or astaxanthinproduction production, the method comprisingsuppressing Tla1 gene expression in a plante to be used for H₂production; and cultivating the plant under conditions in which H₂ isproduced.